Thermoreversible compositions for administration of therapeutic agents

ABSTRACT

The present invention relates to compositions and methods of inducing the self-renewal of stem/progenitor supporting cells, including inducing the stem/progenitor cells to proliferate while maintaining, in the daughter cells, the capacity to differentiate into hair cells.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/239,786, filed Jan. 4, 2019, which is a divisional application ofU.S. application Ser. No. 15/447,682, filed on Mar. 2, 2017, now U.S.Pat. No. 10,213,511, which claims the benefit of and priority to U.S.Provisional Application No. 62/302,747, filed Mar. 2, 2016, the entirecontents of each of which are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

The present invention relates to thermoreversible compositions ofpoloxamers and methods of using same for administration of therapeuticagents

BACKGROUND OF THE INVENTION

Poloxamers are nonionic triblock copolymers composed of a centralhydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked bytwo hydrophilic chains of polyoxyethylene (poly(ethylene oxide)).Poloxamers are often considered “functional excipients” because they areessential components and play an important role in a formulation.

Poloxamers are synthetic triblock co-polymers with the following generalstructure formula:

wherein a=2-130 and b=15-67

Non-limiting examples of poloxamers include: poloxamer 124, poloxamer184, poloxamer 185, poloxamer 188, poloxamer 237, poloxamer 338, andpoloxamer 407. The chemical characteristics of each poloxamer aredetermined by the chain length of the polyethylene (EO) units and thepolyoxypropylene (PO) units, molecular weight and degree ofhydrophobicity/hydrophilicity. Some poloxamers are liquid at roomtemperature (124) whereas others are a solid (184, 185, 188, 237, 338,and 407). Poloxamers have a variety of uses including as emulsifyingagents, solubilizing agents and stabilizing agents. For example,poloxamer 188 has been used as an emulsifying agent for fluorocarbonsused as artificial blood substitutes and in the preparation ofsolid-dispersions. They are also used as wetting agents in ointments,suppository bases, and gels, as well as tablet binders and coatings. Insome cases, poloxamers have been used for solubilizing formulationscontaining otic therapeutic agents and their delivering. In some cases,a poloxamer formulation is created containing active agents that isliquid when at 4° C. which gels when heated at or above roomtemperature. This enables the formulation to be injected through a smallneedle and gel when it hits the middle ear. In these cases the amountsof poloxamer used for solubilization was about 15-17 wt %. Atconcentrations above 15-17 wt %, poloxamers will typically form gelsthat are not flowable even with cooled to 4° C., making them unsuitablefor injection. There is a need for injectable formulations oftherapeutic agents containing a poloxamer with gelation temperatures ofat least 10 degrees or at least body temperature, where the poloxamer isat concentrations higher than about 15-17 wt %.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides a poloxamer containingpharmaceutical composition having a gelation temperature greater than10° C., the composition comprising:

a) a pharmaceutically active therapeutic agent or a pharmaceuticallyacceptable salt thereof;

b) a poloxamer at greater than or equal to 21 wt % of the pharmaceuticalcomposition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition has a gelation temperature ofgreater than 10° C.

In some embodiments, the poloxamer comprises at least one of Poloxamer124, Poloxamer 188, Poloxamer 237, Poloxamer 338 or Poloxamer 407. Insome embodiments, the poloxamer comprises a mixture of two or more ofPoloxamer 124, Poloxamer 188, Poloxamer 237, Poloxamer 338 or Poloxamer407. In some embodiments, the mixture of two or more poloxamerscomprises Poloxamer 407 and Poloxamer 124. In another embodiment, thePoloxamer comprises at least one of Poloxamer 188 and Poloxamer 407 ormixtures thereof. In some embodiments, the poloxamer is Poloxamer 407.

In some embodiments, the poloxamer can be in a concentration between 21wt % and 40 wt % relative to the composition. In another embodiment, thepoloxamer is in a concentration between 21 wt % and 30 wt % relative tothe composition. In another embodiment, the poloxamer is in aconcentration between 23 wt % and 29 wt % relative to the composition.In another embodiment, the poloxamer is in a concentration between 23 wt% and 27 wt % relative to the composition. In another embodiment, thepoloxamer is in a concentration of 25 wt % relative to the composition.

In some embodiments, the gelation temperature of the pharmaceuticalcomposition is between about 11° C. and about 32° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis between is between about 15° C. and about 30° C. In some embodiments,the gelation temperature of the pharmaceutical composition is betweenabout 20° C. and about 28° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is between about 24° C.and about 26° C.

In some embodiments, the gelation temperature of the pharmaceuticalcomposition is about 15° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is about 20° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis about 24° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 26° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 28° C.In some embodiments, the gelation temperature of the pharmaceuticalcomposition is about 30° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the carboxylic acid containingcompound is a substituted or unsubstituted C₅-C₉ straight or branchedchain alkyl-CO₂H.

In any of the foregoing embodiments, the carboxylic acid containingcompound is a substituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is asubstituted C₅-C₉ straight or branched chain alkyl-CO₂H, wherein thesubstituent is —NH₂. In some examples the carboxylic acid containingcompound is an amino substituted 2-propylpentanoic acid. In someembodiments, the amino substituted 2-propylpentanoic acid is selectedfrom the group consisting of 5-amino-2-propylpentanoic acid,4-amino-2-propylpentanoic acid, 3-amino-2-propylpentanoic acid, and2-amino-2-propylpentanoic acid.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₆-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₅-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the unsubstituted C₉ branched straight chainalkyl-CO₂H carboxylic acid containing compound is Valproic acid.

In some embodiments, the Valproic acid can be in the form of an amide.In some embodiments, the amide form of Valproic acid is the condensationproduct with an amino acid. In some embodiments, the amide of Valproicacid is selected from the group consisting of

In any of the foregoing embodiments, the amount of the carboxylic acidcontaining compound in the pharmaceutical composition is between atleast 2 wt % (weight carboxylic acid containing compound/weightpharmaceutical composition) and 20 wt % (weight carboxylic acidcontaining compound/weight pharmaceutical composition). In someembodiments, the composition comprises at least 4 wt % carboxylic acid.In other embodiments, the composition comprises at least 8 wt %carboxylic acid. In some embodiments, the composition comprises at least12 wt % carboxylic acid. In some embodiments, the composition comprisesat least 16 wt % carboxylic acid. In still other embodiments, thecomposition comprises at least 20 wt % carboxylic acid.

In other embodiments, the composition comprises about 4 wt % carboxylicacid. In other embodiments, the composition comprises about 8 wt %carboxylic acid. In other embodiments, the composition comprises about12 wt % carboxylic acid. In further embodiments, the compositioncomprises about 16 wt % carboxylic acid. In further embodiments, thecomposition comprises about 20 wt % carboxylic acid.

In some embodiments of the poloxamer containing pharmaceuticalcomposition, the pharmaceutically active therapeutic agent is selectedfrom the group consisting of a wnt agonist, a GSK3 inhibitor, a2-pyrimidinylaminoethylamino-2-pyridine containing compound, aN-(1H-pyrazol-4-yl)-nicotinamide containing compound, and a1,2,3,4-tetrahydro-[1,4]diazepino[6,7,]indol-7-yl)-4-(imidazo[1,2-a]pyridin-3-yl)-1H-pyrrole-2,5-dionecontaining compound.

A further aspect of the present disclosure provides a poloxamercontaining pharmaceutical composition having a gelation temperaturegreater than 10° C., the composition comprising:

a) a wnt agonist or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 at greater than or equal to 21 wt % of thepharmaceutical composition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition has a gelation temperature ofgreater than 10° C.

A further aspect of the present disclosure provides a poloxamercontaining pharmaceutical composition having a gelation temperaturegreater than 10° C., the composition comprising:

a) a GSK3 inhibitor or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 at greater than or equal to 21 wt % of thepharmaceutical composition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition has a gelation temperature ofgreater than 10° C.

A further aspect of the present disclosure provides a poloxamercontaining pharmaceutical composition having a gelation temperaturegreater than 10° C., the composition comprising

a) a 2-pyrimidinylaminoethylamino-2-pyridine compound;

b) Poloxamer 407 at greater than or equal to 21 wt % of thepharmaceutical composition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition has a gelation temperature ofgreater than 10° C.

A further aspect of the present disclosure provides poloxamer containingpharmaceutical composition having a gelation temperature greater than10° C., the composition comprising:

a) an N-(alkylcarbamoyl)-1H-pyrazol-4-yl)-nicotinamide containingcompound or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 at greater than or equal to 21 wt % of thepharmaceutical composition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition has a gelation temperature ofgreater than 10° C.

A further aspect of the present disclosure provides a poloxamercontaining pharmaceutical composition having a gelation temperaturegreater than 10° C., the composition comprising:

a)1,2,3,4-tetrahydro-[1,4]diazepino[6,7,]indol-7-yl)-1H-pyrrole-2,5-dionecontaining compound or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 at greater than or equal to 21 wt % of thepharmaceutical composition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition has a gelation temperature ofgreater than 10° C.

A further aspect of the present disclosure provides a poloxamercontaining pharmaceutical composition having a gelation temperaturegreater than 10° C., the composition comprising:

a) a compound of Formula I:

or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 in weight percentage of more than 21% wt; and

c) Valproic acid;

wherein the amount of Valproic acid in the pharmaceutical composition isbetween at least 2 wt % (weight carboxylic acid containingcompound/weight pharmaceutical composition) and 20 wt % (weightcarboxylic acid containing compound/weight pharmaceutical composition).In some embodiments, the composition comprises at least 4 wt % Valproicacid. In some embodiments, the composition comprises at least 8 wt %Valproic acid. In other embodiments, the composition comprises at least12 wt % Valproic acid. In some embodiments, the composition comprises atleast 16 wt % Valproic acid. In other embodiments, the compositioncomprises at least 20 wt % Valproic acid.

In other embodiments, the composition comprising Formula I, comprisesbetween about 2 wt % Valproic acid and about 20 wt %. In otherembodiments, the composition comprising Formula I, comprises about 2 wt% Valproic acid. In other embodiments, the composition comprisingFormula I, comprises about 4 wt % Valproic acid. In other embodiments,the composition comprising Formula I, comprises about 8 wt % Valproicacid. In other embodiments, the composition comprising Formula I,comprises about 12 wt % Valproic acid. In further embodiments, thecomposition comprising Formula I, comprises about 16 wt % Valproic acid.In further embodiments, the composition comprising Formula I, comprisesabout 20 wt % Valproic acid.

A further aspect of the present disclosure provides a poloxamercontaining pharmaceutical composition having a gelation temperaturegreater than 10° C., the composition comprising:

a) a compound of Formula II

or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 in weight percentage of more than 21% wt; and

c) Valproic acid;

wherein the amount of Valproic acid in the pharmaceutical composition isbetween at least 2 wt % (weight carboxylic acid containingcompound/weight pharmaceutical composition) and 20 wt %. In someembodiments, the composition comprising the compound of Formula II,comprises at least 4 wt % Valproic acid. In some embodiments, thecomposition comprising the compound of Formula II, comprises at least 8wt % Valproic acid. In other embodiments, the composition comprising thecompound of Formula II, comprises at least 12 wt % Valproic acid. Insome embodiments, the composition comprising the compound of Formula II,comprises at least 16 wt % Valproic acid. In other embodiments, thecomposition comprising the compound of Formula II, comprises at least 20wt % Valproic acid.

In other embodiments, the composition comprising Formula II, comprisesbetween about 2 wt % Valproic acid and about 20 wt %. In otherembodiments, the composition comprising Formula II, comprises about 2 wt% Valproic acid. In other embodiments, the composition comprisingFormula II, comprises about 4 wt % Valproic acid. In other embodiments,the composition comprising Formula II, comprises about 8 wt % Valproicacid. In other embodiments, the composition comprising Formula II,comprises about 12 wt % Valproic acid. In further embodiments, thecomposition comprising Formula II, comprises about 16 wt % Valproicacid. In further embodiments, the composition comprising Formula II,comprises about 20 wt % Valproic acid.

A further aspect of the present disclosure provides a poloxamercontaining pharmaceutical composition having a gelation temperaturegreater than 10° C., the composition comprising:

a) a compound of Formula III

or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 in weight percentage of more than 21% wt; and

c) Valproic acid;

wherein the amount of Valproic acid in the pharmaceutical composition isbetween at least 2 wt % (weight carboxylic acid containingcompound/weight pharmaceutical composition) and 20 wt %.

In some embodiments, the composition comprising the compound of FormulaIII, comprises at least 4 wt % Valproic acid. In some embodiments, thecomposition comprising the compound of Formula III, comprises at least 8wt % Valproic acid. In other embodiments, the composition comprising thecompound of Formula III, comprises at least 12 wt % Valproic acid. Insome embodiments, the composition comprising the compound of FormulaIII, comprises at least 16 wt % Valproic acid. In other embodiments, thecomposition comprising the compound of Formula III, comprises at least20 wt % Valproic acid.

In other embodiments, the composition comprising Formula III, comprisesbetween about 2 wt % Valproic acid and about 20 wt %. In otherembodiments, the composition comprising Formula III, comprises about 2wt % Valproic acid. In other embodiments, the composition comprisingFormula III, comprises about 4 wt % Valproic acid. In other embodiments,the composition comprising Formula III, comprises about 8 wt % Valproicacid. In other embodiments, the composition comprising Formula III,comprises about 12 wt % Valproic acid. In further embodiments, thecomposition comprising Formula III, comprises about 16 wt % Valproicacid. In further embodiments, the composition comprising Formula III,comprises about 20 wt % Valproic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing the solubility of Poloxamer 407 (P407) at16.7% of w/w (16.7 grams Poloxamer 407 to 83.3 grams of a solution thatcontains % 1 Molar valproic acid sodium salt/5% weight DMSO and water),28.6% w/w, 33.3% w/w, 37.5% w/w, 41.2% w/w, and 50.0% w/w in 1M Valproicacid sodium salt and 5% dimethylsulfoxide (DMSO) in water. Percent w/wof Poloxamer 407 left to right as shown: 16.7%, 28.6%, 33.3%, 37.5%,41.2%, and 50.0%.

FIG. 2 is a photograph showing the solubility of Poloxamer 407 (P407) at20% w/w, 23.1% w/w, and 25.9% w/w Poloxamer 407 in 1M Valproic acidsodium salt and 5% dimethylsulfoxide (DMSO). Percent w/w of P407 left toright as shown: 20% w/w, 23.1% w/w, and 25.9% w/w.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure provides poloxamer containing pharmaceuticalcompositions which exhibit a gelation temperature greater than 10° C.These compositions comprise pharmaceutically active therapeutic agentsor their pharmaceutically acceptable salts. Moreover, thesepharmaceutical compositions comprise both poloxamer at greater than orequal to 21 wt % of the pharmaceutical composition and a carboxylic acidcontaining compound and have a gelation temperature of greater than 10°C. The carboxylic acid containing compound is in an amount of between atleast 2 wt % and 20 wt %. These compositions are described herein aswell as some non-limiting uses of same.

Definitions

In this application, the use of “or” means “and/or” unless statedotherwise. As used in this application, the term “comprise” andvariations of the term, such as “comprising” and “comprises,” are notintended to exclude other additives, components, integers or steps. Asused in this application, the terms “about” and “approximately” are usedas equivalents. Any numerals used in this application with or withoutabout/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art. In certainembodiments, the term “approximately” or “about” refers to a range ofvalues that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in eitherdirection (greater than or less than) of the stated reference valueunless otherwise stated or otherwise evident from the context (exceptwhere such number would exceed 100% of a possible value).

“Administration” refers to introducing a substance into a subject. Insome embodiments, administration is auricular, intraauricular,intracochlear, intravestibular, or transtympanically, e.g., byinjection. In some embodiments, administration is directly to the innerear, e.g., injection through the round or oval window, otic capsule, orvestibular canals. In some embodiments, administration is directly intothe inner ear via a cochlear implant delivery system. In someembodiments, the substance is injected transtympanically to the middleear. In certain embodiments “causing to be administered” refers toadministration of a second component after a first component has alreadybeen administered (e.g., at a different time and/or by a differentactor).

An “antibody” refers to an immunoglobulin polypeptide, or fragmentthereof, having immunogen binding ability.

As used herein, an “agonist” is an agent that causes an increase in theexpression or activity of a target gene, protein, or a pathway,respectively. Therefore, an agonist can bind to and activate its cognatereceptor in some fashion, which directly or indirectly brings about thisphysiological effect on the target gene or protein. An agonist can alsoincrease the activity of a pathway through modulating the activity ofpathway components, for example, through inhibiting the activity ofnegative regulators of a pathway. Therefore, a “Wnt agonist” can bedefined as an agent that increases the activity of Wnt pathway, whichcan be measured by increased TCF/LEF-mediated transcription in a cell.Therefore, a “Wnt agonist” can be a true Wnt agonist that binds andactivates a Frizzled receptor family member, including any and all ofthe Wnt family proteins, an inhibitor of intracellular beta-catenindegradation, and activators of TCF/LEF.

An “antagonist” refers to an agent that binds to a receptor, and whichin turn decreases or eliminates binding by other molecules.

“Anti-sense” refers to a nucleic acid sequence, regardless of length,that is complementary to the coding strand or mRNA of a nucleic acidsequence. Antisense RNA can be introduced to an individual cell, tissueor organanoid. An anti-sense nucleic acid can contain a modifiedbackbone, for example, phosphorothioate, phosphorodithioate, or othermodified backbones known in the art, or may contain non-naturalinternucleoside linkages.

As referred to herein, a “complementary nucleic acid sequence” is anucleic acid sequence capable of hybridizing with another nucleic acidsequence comprised of complementary nucleotide base pairs. By“hybridize” is meant pair to form a double-stranded molecule betweencomplementary nucleotide bases (e.g., adenine (A) forms a base pair withthymine (T), as does guanine (G) with cytosine (C) in DNA) undersuitable conditions of stringency. (See, e.g., Wahl, G. M. and S. L.Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) MethodsEnzymol. 152:507).

“Auricular administration” refers to a method of using a catheter orwick device to administer a composition across the tympanic membrane tothe inner ear of the subject. To facilitate insertion of the wick orcatheter, the tympanic membrane may be pierced using a suitably sizedsyringe or pipette. The devices could also be inserted using any othermethods known to those of skill in the art, e.g., surgical implantationof the device. In particular embodiments, the wick or catheter devicemay be a stand-alone device, meaning that it is inserted into the ear ofthe subject and then the composition is controllably released to theinner ear. In other particular embodiments, the wick or catheter devicemay be attached or coupled to a pump or other device that allows for theadministration of additional compositions. The pump may be automaticallyprogrammed to deliver dosage units or may be controlled by the subjector medical professional.

“Biocompatible Matrix” as used herein is a polymeric carrier that isacceptable for administration to humans for the release of therapeuticagents. A Biocompatible Matrix may be a biocompatible gel or foam.

“Cell Aggregate” as used herein shall mean a body cells in the Organ ofCorti that have proliferated to form a cluster of a given cell type thatis greater than 40 microns in diameter and/or produced a morphology inwhich greater than 3 cell layers reside perpendicular to the basilarmembrane. A “Cell Aggregate” can also refer a process in which celldivision creates a body of cells that cause one or more cell types tobreach the reticular lamina, or the boundary between endolymph andperilymph

“Cell Density” as used herein in connection with a specific cell type isthe mean number of that cell type per area in a RepresentativeMicroscopy Sample. The cell types may include but are not limited toLgr5⁺ cells, hair cells, or supporting cells. The Cell Density may beassessed with a given cell type in a given organ or tissue, includingbut not limited to the cochlea or Organ of Corti. For instance, theLgr5+ Cell Density in the Organ of Corti is the Cell Density of Lgr5⁺cells as measured across the Organ of Corti. Typically, supporting cellsand Lgr5⁺ cells will be enumerated by taking cross sections of the Organof Corti. Typically, hair cells will be enumerated by looking down atthe surface of the Organ of Corti, though cross sections may be used insome instances, as described in a Representative Microscopy Sample.Typically, Cell Density of Lgr5+ cells will be measured by analyzingwhole mount preparations of the Organ of Corti and counting the numberof Lgr5 cells across a given distance along the surface of theepithelia, as described in a Representative Microscopy Sample. Haircells may be identified by their morphological features such as bundlesor hair cell specific stains (e.g., Myosin VIIa, Prestin, vGlut3,Pou4f3, Espin, conjugated-Phalloidin, PMCA2, Ribeye, Atoh1, etc). Lgr5+cells may be identified by specific stains or antibodies (e.g., Lgr5-GFPtransgenic reporter, anti-Lgr5 antibody, etc.)

“Cochlear Concentration” as used herein will be the concentration of agiven agent as measured through sampling cochlear fluid. Unlessotherwise noted, the sample should contain a substantial enough portionof the cochlear fluid so that it is approximately representative of theaverage concentration of the agent in the cochlea. For example, samplesmay be drawn from a vestibular canal, and a series of fluid samplesdrawn in series such that individual samples are comprised of cochlearfluid in specified portions of the cochlea

“Complementary nucleic acid sequence” refers to a nucleic acid sequencecapable of hybridizing with another nucleic acid sequence comprised ofcomplementary nucleotide base pairs.

“Cross-Sectional Cell Density” as used herein in connection with aspecific cell type is the mean number of that cell type per area ofcross section through a tissue in a Representative Microscopy Sample.Cross sections of the Organ of Corti can also be used to determine thenumber of cells in a given plane. Typically, hair cells Cross-sectionalCell Density will be measured by analyzing whole mount preparations ofthe Organ of Corti and counting the number of hair cells across a givendistance in cross sections taken along a portion of the epithelia, asdescribed in a Representative Microscopy Sample. Typically,Cross-sectional Cell Density of Lgr5⁺ cells will be measured byanalyzing whole mount preparations of the Organ of Corti and countingthe number of Lgr5⁺ cells across a given distance in cross sectionstaken along a portion of the epithelia, as described in a RepresentativeMicroscopy Sample. Hair cells may be identified by their morphologicalfeatures such as bundles or hair cell specific stains (suitable stainsinclude e.g., Myosin VIIa, Prestin, vGlut3, Pou4f3,conjugated-Phalloidin, PMCA2, Atoh1, etc.). Lgr5⁺ cells may beidentified by specific stains or antibodies (suitable stains andantibodies include fluorescence in situ hybridization of Lgr5 mRNA,Lgr5-GFP transgenic reporter system, anti-Lgr5 antibodies, etc.).

“Decreasing” refers to decreasing by at least 5%, for example, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 99 or 100%, for example, as compared to the level of reference.

“Decreases” also means decreases by at least 1-fold, for example, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,500, 1000-fold or more, for example, as compared to the level of areference.

“Differentiation Period” as used herein is the duration of time in whichthere is an Effective Stemness Driver Concentration

“Effective Concentration” may be the Effective Stemness DriverConcentration for a Stemness Driver.

“Effective Release Rate” (mass/time) as used herein is the EffectiveConcentration (mass/volume)*30 uL/1 hour.

“Effective Stemness Driver Concentration” is the minimum concentrationof a Stemness Driver that induces at least 1.5-fold increase in numberof LGR5+ cells in a Stem Cell Proliferation Assay compared to the numberof Lgr5+ cells in a Stem Cell Proliferation Assay performed without theStemness Driver and with all other components present at the sameconcentrations.

“Eliminate” means to decrease to a level that is undetectable.

“Engraft” or “engraftment” refers to the process of stem or progenitorcell incorporation into a tissue of interest in vivo through contactwith existing cells of the tissue. “Epithelial progenitor cell” refersto a multipotent cell which has the potential to become restricted tocell lineages resulting in epithelial cells.

“Epithelial stem cell” refers to a multipotent cell which has thepotential to become committed to multiple cell lineages, including celllineages resulting in epithelial cells.

“Fragment” refers to a portion of a polypeptide or nucleic acidmolecule. This portion contains, at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90% of the entire length of the reference nucleic acidmolecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60,70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000nucleotides or amino acids.

“Gelation Temperature,” as used herein is defined as meaning thetemperature below which a composition is in liquid form and above whichthe composition forms a semi-solid, for example, a gel.

“GSK3beta,” “GSK3β,” and “GSK3B” as used interchangeably herein areacronyms for glycogen synthase kinase 3 beta,

“GSK3 inhibitor” is a composition that inhibits the activity of anyGSK3.

“GSK3beta inhibitor” is a composition that inhibits the activity ofGSK3beta.

“GSK3alpha,” “GSK3α,” and “GSK3A” as used interchangeably herein areacronyms for glycogen synthase kinase 3 alpha,

“GSK3alpha inhibitor” is a composition that inhibits the activity ofGSK3alpha.

“Hybridize” refers to pairing to form a double-stranded molecule betweencomplementary nucleotide bases (e.g., adenine (A) forms a base pair withthymine (T), as does guanine (G) with cytosine (C) in DNA) undersuitable conditions of stringency. (See, e.g., Wahl, G. M. and S. L.Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) MethodsEnzymol. 152:507).

An “inhibitor” refers to an agent that causes a decrease in theexpression or activity of a target gene or protein, respectively. An“antagonist” can be an inhibitor, but is more specifically an agent thatbinds to a receptor, and which in turn decreases or eliminates bindingby other molecules.

As used herein, an “inhibitory nucleic acid” is a double-stranded RNA,RNA interference, miRNA, siRNA, shRNA, or antisense RNA, or a portionthereof, or a mimetic thereof, that when administered to a mammaliancell results in a decrease in the expression of a target gene.Typically, a nucleic acid inhibitor comprises at least a portion of atarget nucleic acid molecule, or an ortholog thereof, or comprises atleast a portion of the complementary strand of a target nucleic acidmolecule. Typically, expression of a target gene is reduced by 10%, 25%,50%, 75%, or even 90-100%.

“In vitro Lgr5 activity” refers to the level of expression or activityof Lgr5 in an in vitro population of cells. It may be measured, forexample, in cells derived from a Lgr5-GFP expressing mouse such as aB6.129P2-Lgr5tm1(cre/ERT2)Cle/J mouse (also known asLgr5-EGFP-IRES-creERT2 or Lgr5-GFP mouse, Jackson Lab Stock No: 008875)by dissociating cells to single cells, staining with propidium iodide(PI), and analyzing the cells using a flow cytometer for Lgr5-GFPexpression. Inner ear epithelial cells from wild-type (non-Lgr5-GFP)mice that passing the same culturing and analyzing procedures can beused as a negative control. Typically, two populations of cells areshown in the bivariate plot with GFP/FITC as one variable, which includeboth GFP positive and GFP negative populations. Lgr5-positive cells areidentified by gating GFP positive cell population. The percentage ofLgr5-positive cells is measured by gating GFP positive cell populationagainst both GFP negative population and the negative control. Thenumber of Lgr5-positive cells is calculated by multiplying the totalnumber of cells by the percentage of Lgr5-positive cells. For cellsderived from non-Lgr5-GFP mice, Lgr5 activity can be measured using ananti-Lgr5 antibody or quantitative-PCR on the Lgr5 gene.

“In vivo Lgr5 activity” as used herein is the level of expression oractivity of Lgr5 in a subject. It may be measured, for example, byremoving an animal's inner ear and measuring Lgr5 protein or Lgr5 mRNA.Lgr5 protein production can be measured using an anti-Lgr5 antibody tomeasure fluorescence intensity as determined by imaging cochlearsamples, where fluorescence intensity is used as a measure of Lgr5presence. Western blots can be used with an anti-Lgr5 antibody, wherecells can be harvested from the treated organ to determine increases inLgr5 protein. Quantitative-PCR or RNA in situ hybridization can be usedto measure relative changes in Lgr5 mRNA production, where cells can beharvested from the inner ear to determine changes in Lgr5 mRNA.Alternatively, Lgr5 expression can be measured using an Lgr5 promoterdriven GFP reporter transgenic system, where the presence or intensityGFP fluoresce can be directly detected using flow cytometry, imaging, orindirectly using an anti-GFP antibody.

“Increases” also means increases by at least 1-fold, for example, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,500, 1000-fold or more, for example, as compared to the level of a ascompared to the level of a reference standard.

“Increasing” refers to increasing by at least 5%, for example, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 99, 100% or more, for example, as compared to the level of areference.

“Intraauricular administration” refers to administration of acomposition to the middle or inner ear of a subject by directlyinjecting the composition.

“Intracochlear” administration refers to direct injection of acomposition across the tympanic membrane and across the round or ovalwindow membrane into the cochlea.

“Intravestibular” administration refers to direct injection of acomposition across the tympanic membrane and across the round or ovalwindow membrane into the vestibular organs.

“Isolated” refers to a material that is free to varying degrees fromcomponents which normally accompany it as found in its native state.“Isolate” denotes a degree of separation from original source orsurroundings.

“Lgr5” is an acronym for the Leucine-rich repeat-containing G-proteincoupled receptor 5, also known as G-protein coupled receptor 49 (GPR49)or G-protein coupled receptor 67 (GPR67). It is a protein that in humansis encoded by the Lgr5 gene.

“Lgr5 activity” is defined as the level of activity of Lgr5 in apopulation of cells. In an in vitro cell population, Lgr5 activity maybe measured in an in vitro Lgr5 Activity assay. In an in vivo cellpopulation, Lgr5 activity may be measured in an in vivo Lgr5 Activityassay.

“Lgr5⁺ cell” or “Lgr5-positive cell” as used herein is a cell thatexpresses Lgr5. “Lgr5⁻ cell” as used herein is a cell that is not Lgr5⁺.

“Lineage Tracing” as used herein is using a mouse line that enables fatetracing of any cell that expresses a target gene at the time of reporterinduction. This can include hair cell or supporting cells genes (Sox2,Lgr5, MyosinVIIa, Pou4f3, etc). For example, lineage tracing may use anLgr5-EGFP-IRES-creERT2 mouse crossed with a reporter mouse, which uponinduction, allows one to trace the fate of cells that expressed Lgr5 atthe time of induction. By further example, Lgr5 cells can be isolatedinto single cells and cultured in a Stem Cell Proliferation Assay togenerate colonies, then subsequently differentiated in a DifferentiationAssay and analyzed for cell fate by staining for hair cell and/orsupporting cell proteins and determining the reporter colocalizationwith either hair cell or supporting cell staining to determine the Lgr5cells' fate. In addition, lineage tracing can be performed in cochlearexplants to track supporting cell or hair cell fate within the intactorgan after treatment. For example, Lgr5 cell fate can be determined byisolating the cochlea from a Lgr5-EGFP-IRES-creERT2 mouse crossed with areporter mouse, and inducing the reporter in Lgr5 cells before or duringtreatment. The organ can then be analyzed for cell fate by staining forhair cell and/or supporting cell proteins and determining the reportercolocalization with either hair cell or supporting cell staining todetermine the Lgr5 cells' fate. In addition, lineage tracing can beperformed in vivo track supporting cell or hair cell fate within theintact organ after treatment. For example, Lgr5 cell fate can bedetermined inducing a reporter in an Lgr5-EGFP-IRES-creERT2 mousecrossed with a reporter mouse, treating the animal, then isolating thecochlea. The organ can then be analyzed for cell fate by staining forhair cell and/or supporting cell proteins and determining the reportercolocalization with either hair cell or supporting cell staining todetermine the Lgr5 cells' fate. Lineage tracing may be performed usingalternative reporters of interest as is standard in the art.

“Mammal” refers to any mammal including but not limited to human, mouse,rat, sheep, monkey, goat, rabbit, hamster, horse, cow or pig.

“Mean Release Time” as used herein is the time in which one-half of anagent is released into phosphate buffered saline from a carrier in aRelease Assay.

“Native Morphology” as used herein is means that tissue organizationlargely reflects the organization in a healthy tissue.

“Non-human mammal”, as used herein, refers to any mammal that is not ahuman.

As used in relevant context herein, the term “number” of cells can be 0,1, or more cells.

“Organ of Corti” as used herein refers to the sensory cells (inner andouter hair cells) of the hearing organ located in the cochlea.

“Organoid” or “epithelial organoid” refers to a cell cluster oraggregate that resembles an organ, or part of an organ, and possessescell types relevant to that particular organ.

“Population” of cells refers to any number of cells greater than 1, butis at least 1×10³ cells, at least 1×10⁴ cells, at least at least 1×10⁵cells, at least 1×10⁶ cells, at least 1×10⁷ cells, at least 1×10⁸ cells,at least 1×10⁹ cells, or at least 1×10¹⁰ cells.

“Progenitor cell” as used herein refers to a cell that, like a stemcell, has the tendency to differentiate into a specific type of cell,but is already more specific than a stem cell and is pushed todifferentiate into its “target” cell.

“Proliferation Period” as used herein is the duration of time in whichthere is an Effective Stemness Driver Concentration.

“Reference” means a standard or control condition (e.g., untreated witha test agent or combination of test agents).

“Release Assay” as used herein is a test in which the rate of release ofan agent from a Biocompatible Matrix through dialysis membrane to asaline environment. An exemplary Release Assay may be performed byplacing 30 microliters of a composition in 1 ml Phosphate BufferedSaline inside saline dialysis bag with a suitable cutoff, and placingthe dialysis bag within 10 mL of Phosphate Buffered Saline at 37 C. Thedialysis membrane size may be chosen based on agent size in order toallow the agent being assessed to exit the membrane. For small moleculerelease, a 3.5-5 kDa cutoff may be used. The Release Rate for acomposition may change over time and may be measured in 1 hourincrements.

“Representative Microscopy Sample” as used herein describes a sufficientnumber of fields of view within a cell culture system, a portion ofextracted tissue, or an entire extracted organ that the average featuresize or number being measured can reasonably be said to represent theaverage feature size or number if all relevant fields were measured. Forexample, in order to assess the hair cell counts at a frequency range onthe Organ of Corti, ImageJ software (NIH) can used to measure the totallength of cochlear whole mounts and the length of individual countedsegments. The total number of inner hair cells, outer hair cells, andsupporting cells can be counted in the entire or fraction of any of thefour cochlear segments of 1200-1400 μm (apical, mid-apical, mid-basal,and basal) at least 3 fields of view at 100 μm field size would bereasonably considered a Representative Microscopy Sample. ARepresentative Microscopy sample can include measurements within a fieldof view, which can be measured as cells per a given distance. ARepresentative Microscopy sample can be used to assess morphology, suchas cell-cell contacts, cochlear architecture, and cellular components(e.g., bundles, synapses).

“Rosette Patterning” is a characteristic cell arrangement in the cochleain which <5% hair cells are adjacent to other hair cells.

The term “sample” refers to a volume or mass obtained, provided, and/orsubjected to analysis. In some embodiments, a sample is or comprises atissue sample, cell sample, a fluid sample, and the like. In someembodiments, a sample is taken from (or is) a subject (e.g., a human oranimal subject). In some embodiments, a tissue sample is or comprisesbrain, hair (including roots), buccal swabs, blood, saliva, semen,muscle, or from any internal organs, or cancer, precancerous, or tumorcells associated with any one of these. A fluid may be, but is notlimited to, urine, blood, ascites, pleural fluid, spinal fluid, and thelike. A body tissue can include, but is not limited to, brain, skin,muscle, endometrial, uterine, and cervical tissue or cancer,precancerous, or tumor cells associated with any one of these. In anembodiment, a body tissue is brain tissue or a brain tumor or cancer.Those of ordinary skill in the art will appreciate that, in someembodiments, a “sample” is a “primary sample” in that it is obtainedfrom a source (e.g., a subject); in some embodiments, a “sample” is theresult of processing of a primary sample, for example to remove certainpotentially contaminating components and/or to isolate or purify certaincomponents of interest.

“Self-renewal” refers to the process by which a stem cell divides togenerate one (asymmetric division) or two (symmetric division) daughtercells with development potentials that are indistinguishable from thoseof the mother cell. Self-renewal involves both proliferation and themaintenance of an undifferentiated state.

“siRNA” refers to a double stranded RNA. Optimally, an siRNA is 18, 19,20, 21, 22, 23 or 24 nucleotides in length and has a 2 base overhang atits 3′ end. These dsRNAs can be introduced to an individual cell orculture system. Such siRNAs are used to downregulate mRNA levels orpromoter activity.

“Stem cell” refers to a multipotent cell having the capacity toself-renew and to differentiate into multiple cell lineages.

“Stem Cell Differentiation Assay” as used herein is an assay todetermine the differentiation capacity of stem cells. In an exemplaryStem Cell Differentiation Assay, the number of cells for an initial cellpopulation is harvested from a Atoh1-GFP mouse between the age of 3 to 7days, by isolating the Organ of Corti sensory epithelium, dissociatingthe epithelium into single cells, and passing the cells through a 40 umcell strainer. Approximately 5000 cells are entrapped in 40 μl ofculture substrate (for example: Matrigel (Corning, Growth FactorReduced)) and placed at the center of wells in a 24-well plate with 500μl of an appropriate culture media, growth factors and agent beingtested. Appropriate culture media and growth factors include AdvancedDMEM/F12 with media Supplements (1× N2, 1× B27, 2 mM Glutamax, 10 mMHEPES, 1 mM N-acetylcysteine, and 100 U/ml Penicillin/100 μg/mlStreptomycin) and growth factors (50 ng/ml EGF, 50 ng/ml bFGF, and 50ng/ml IGF-1) as well as the agent(s) being assessed are added into eachwell. Cells are cultured for 10 days in a standard cell cultureincubator at 37° C. and 5% CO₂, with media change every 2 days. Thesecells are then cultured by removing the Stem Cell Proliferation Assayagents and replacing with Basal culture media and molecules to drivedifferentiation. An appropriate Basal culture media is Advanced DMEM/F12supplemented with 1× N2, 1× B27, 2 mM Glutamax, 10 mM HEPES, 1 mMN-acetylcysteine, and 100 U/ml Penicillin/100 μg/ml Streptomycin andappropriate molecules to drive differentiation are 3 μM CHIR99021 and 5μM DAPT for 10 days, with media change every 2 days. The number of haircells in a population may be measured by using flow cytometry for GFP.Hair cell differentiation level can further be assessed using qPCR tomeasure hair cell marker (e.g., Myo7a) expression level normalized usingsuitable and unregulated references or housekeeping genes (e.g., Hprt).Hair cell differentiation level can also be assessed by immunostainingfor hair cell markers (e.g., Myosin7a, vGlut3, Espin, PMCAs, Ribeye,conjugated-phalloidin, Atoh1, Pou4f3, etc). Hair cell differentiationlevel can also be assessed by Western Blot for Myosin7a, vGlut3, Espin,PMCAs, Prestin, Ribeye, Atoh1, and Pou4f3.

“Stem Cell Assay” as used herein is an assay in which a cell or a cellpopulation are tested for a series of criteria to determine whether thecell or cell population are stem cells or enriched in stem cells or stemcell markers. In a stem cell assay, the cell/cell populations are testedfor stem cell characteristics such as expression of Stem Cell Markers,and further optionally are tested for stem cell function, including thecapacity of self-renewal and differentiation.

“Stem Cell Proliferator” as used herein is a composition that induces anincrease in a population of cells which have the capacity forself-renewal and differentiation.

“Stem Cell Proliferation Assay” as used herein is an assay to determinethe capacity for agent(s) to induce the creation of stem cells from astarting cell population. In an exemplary Stem Cell Proliferation Assay,the number of cells for an initial cell population is harvested from aLgr5-GFP mouse such as a B6.129P2-Lgr5tm1(cre/ERT2)Cle/J mouse (alsoknown as Lgr5-EGFP-IRES-creERT2 or Lgr5-GFP mouse, Jackson Lab Stock No:008875) between the age of 3 to 7 days, by isolating the Organ of Cortisensory epithelium and dissociating the epithelium into single cells.Approximately 5000 cells are entrapped in 40 μl of culture substrate(for example: Matrigel (Corning, Growth Factor Reduced)) and placed atthe center of wells in a 24-well plate with 500 μl of an appropriateculture media, growth factors and agent being tested. Appropriateculture media and growth factors include Advanced DMEM/F12 with mediaSupplements (1× N2, 1× B27, 2 mM Glutamax, 10 mM HEPES, 1 mMN-acetylcysteine, and 100 U/ml Penicillin/100 μg/ml Streptomycin) andgrowth factors (50 ng/ml EGF, 50 ng/ml bFGF, and 50 ng/ml IGF-1) as wellas the agent(s) being assessed are added into each well. Cells arecultured for 10 days in a standard cell culture incubator at 37° C. and5% CO₂, with media change every 2 days. The number of Lgr5⁺ cells isquantified by counting the number of cells identified as Lgr5+ in an Invitro Lgr5 activity assay. The fraction of cells that are Lgr5⁺ isquantified by dividing the number of cells identified as Lgr5⁺ in a cellpopulation by the total number of cells present in the cell population.The average Lgr5⁺ activity of a population is quantified by measuringthe average mRNA expression level of Lgr5 of the population normalizedusing suitable and unregulated references or housekeeping genes (e.g.,Hprt). The number of hair cells in a population may be measured bystaining with hair cell marker (e.g., Myosin VIIa), or using anendogenous reporter of hair cell genes (e.g., Pou4f3-GFP, Atoh1-nGFP)and analyzing using flow cytometry. The fraction of cells that are haircells is quantified by dividing the number of cells identified as haircells in a cell population by the total number of cells present in thecell population. Lgr5 activity can be measured by qPCR.

“Stem Cell Markers” as used herein can be defined as gene products(e.g., protein, RNA, etc) that specifically expressed in stem cells. Onetype of stem cell marker is gene products that are directly andspecifically support the maintenance of stem cell identity. Examplesinclude Lgr5 and Sox2. Additional stem cell markers can be identifiedusing assays that have been described in the literature. To determinewhether a gene is required for maintenance of stem cell identity,gain-of-function and loss-of-function studies can be used. Ingain-of-function studies, over expression of specific gene product (thestem cell marker) would help maintain the stem cell identity. While inloss-of-function studies, removal of the stem cell marker would causeloss of the stem cell identity or induced the differentiation of stemcells. Another type of stem cell marker is gene that only expressed instem cells but does not necessary to have specific function to maintainthe identity of stem cells. This type of markers can be identified bycomparing the gene expression signature of sorted stem cells andnon-stem cells by assays such as micro-array and qPCR. This type of stemcell marker can be found in the literature. (e.g., Liu Q. et al., Int JBiochem Cell Biol. 2015 March; 60: 99-111.http://www.ncbi.nlm.nih.gov/pubmed/25582750). Potential stem cellmarkers include Ccdc121, Gdf10, Opcm1, Phex, etc. The expression of stemcell markers such as Lgr5 or Sox2 in a given cell or cell population canbe measure using assays such as qPCR, immunohistochemistry, westernblot, and RNA hybridization. The expression of stem cell markers canalso be measured using transgenic cells express reporters which canindicate the expression of the given stem cell markers, e.g., Lgr5-GFPor Sox2-GFP. Flow cytometry analysis can then be used to measure theactivity of reporter expression. Fluorescence microscopy can also beused to directly visualize the expression of reporters. The expressionof stem cell markers may further be determined using microarray analysisfor global gene expression profile analysis. The gene expression profileof a given cell population or purified cell population can be comparedwith the gene expression profile of the stem cell to determinesimilarity between the 2 cell populations. Stem cell function can bemeasured by colony forming assay or sphere forming assay, self-renewalassay and differentiation assay. In colony (or sphere) forming assay,when cultured in appropriate culture media, the stem cell should be ableto form colonies, on cell culture surface (e.g., cell culture dish) orembedded in cell culture substrate (e.g., Matrigel) or be able to formspheres when cultured in suspension. In colony/sphere forming assay,single stem cells are seeded at low cell density in appropriate culturemedia and allowed to proliferate for a given period of time (7-10 days).Colony formed are then counted and scored for stem cell markerexpression as an indicator of stemness of the original cell. Optionally,the colonies that formed are then picked and passaged to test itsself-renewal and differentiation potential. In self-renewal assay, whencultured in appropriate culture media, the cells should maintain stemcell marker (e.g., Lgr5) expression over at least one (e.g., 1, 2, 3, 4,5, 10, 20, etc) cell divisions. In a Stem Cell Differentiation Assay,when cultured in appropriate differentiation media, the cells should beable to generate hair cell which can be identified by hair cell markerexpression measured by qPCR, immunostaining, western blot, RNAhybridization or flow cytometry.

“Stemness Driver” as used herein is a composition that inducesproliferation of LGR5⁺ cells, upregulates Lgr5 in cells, or maintainsLgr5 expression in cells, while maintaining the potential forself-renewal and the potential to differentiate into hair cells.Generally, stemness drivers upregulate at least one biomarker ofpost-natal stem cells. Stemness Drivers include but are not limited toWnt agonists and GSK3Beta inhibitors.

“Subject” includes humans and mammals (e.g., mice, rats, pigs, cats,dogs, and horses). In many embodiments, subjects are mammals,particularly primates, especially humans. In some embodiments, subjectsare livestock such as cattle, sheep, goats, cows, swine, and the like;poultry such as chickens, ducks, geese, turkeys, and the like; anddomesticated animals particularly pets such as dogs and cats. In someembodiments (e.g., particularly in research contexts) subject mammalswill be, for example, rodents (e.g., mice, rats, hamsters), rabbits,primates, or swine such as inbred pigs and the like.

“Supporting Cell” as used herein in connection with a cochlearepithelium comprises epithelial cells within the organ of Corti that arenot hair cells. This includes inner pillar cells, outer pillar cells,inner phalangeal cells, Deiter cells, Hensen cells, Boettcher cells,and/or Claudius cells.

“Synergy” or “synergistic effect” is an effect which is greater than thesum of each of the effects taken separately; a greater than additiveeffect.

“TgfBeta inhibitor” as used herein is a composition that reducesactivity of TgfBeta

“Thermoreversible gel” means a material that reversibly changes from aliquid to a gel with a change in temperature.

“Tissue” is an ensemble of similar cells from the same origin thattogether carries out a specific function including, for example, tissueof cochlear, such as the Organ of Corti.

“Transtympanic” administration refers to direct injection of acomposition across the tympanic membrane into the middle ear.

“Treating” as used herein in connection with a cell population meansdelivering a substance to the population to effect an outcome. In thecase of in vitro populations, the substance may be directly (or evenindirectly) delivered to the population. In the case of in vivopopulations, the substance may be delivered by administration to thehost subject.

“Valproic acid” (VPA) has chemical formula C₈H₁₆O₂ and the followingalternate name: 2-propylpentanoic acid. Its chemical structure is asfollows:

As used herein, the term “Valproic acid” also includes derivatives ofValproic Acid including its salts, amides, prodrugs and esters.

“Wnt activation” as used herein in connection with a composition is anactivation of the Wnt signaling pathway.

The use of “or” means “and/or” unless stated otherwise. As used in thisapplication, the term “comprise” and variations of the term, such as“comprising” and “comprises,” are not intended to exclude otheradditives, components, integers or steps. As used in this application,the terms “about” and “approximately” are used as equivalents. Anynumerals used in this application with or without about/approximatelyare meant to cover any normal fluctuations appreciated by one ofordinary skill in the relevant art. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein “pharmaceutically acceptable carrier, diluent orexcipient” includes without limitation any adjuvant, carrier, excipient,glidant, sweetening agent, diluent, preservative, dye/colorant, flavorenhancer, surfactant, wetting agent, dispersing agent, suspending agent,stabilizer, isotonic agent, solvent, surfactant, or emulsifier which hasbeen approved by the United States Food and Drug Administration as beingacceptable for use in humans or domestic animals. Exemplarypharmaceutically acceptable carriers include, but are not limited to, tosugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate;tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal andvegetable fats, paraffins, silicones, bentonites, silicic acid, zincoxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesameoil, olive oil, corn oil and soybean oil; glycols, such as propyleneglycol; polyols, such as glycerin, sorbitol, mannitol and polyethyleneglycol; esters, such as ethyl oleate and ethyl laurate; agar; bufferingagents, such as magnesium hydroxide and aluminum hydroxide; alginicacid; pyrogen-free water; isotonic saline; Ringer's solution; ethylalcohol; phosphate buffer solutions; and any other compatible substancesemployed in pharmaceutical compositions.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as, but are not limited to,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as, but not limitedto, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid,ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid,4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid,citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,gluconic acid, glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuricacid, isobutyric acid, lactic acid, lactobionic acid, lauric acid,maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonicacid, mucic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, l-hydroxy-2-naphthoic acid, nicotinic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid,4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid,tartaric acid, thiocyanic acid,/toluenesulfonic acid, trifluoroaceticacid, undecylenic acid, and the like.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.For example, inorganic salts include, but are not limited to, ammonium,sodium, potassium, calcium, and magnesium salts. Salts derived fromorganic bases include, but are not limited to, salts of primary,secondary, and tertiary amines, substituted amines including naturallyoccurring substituted amines, cyclic amines and basic ion exchangeresins, such as ammonia, isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Non-limiting examples of organic bases used in certain embodimentsinclude isopropylamine, diethylamine, ethanolamine, trimethylamine,dicyclohexylamine, choline and caffeine.

“w/w” refers to the ratio of reagents in a composition measured usingthe weight of the reagent in grams, milligrams, or kilograms.

“wt %” refers to the percent of that agent in the composition measuredusing the weight of the agent measured in grams, milligrams, orkilograms.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

One aspect the present disclosure provides a poloxamer containingpharmaceutical composition having a gelation temperature greater than10° C., the composition comprising:

a) a pharmaceutically active therapeutic agent or a pharmaceuticallyacceptable salt thereof;

b) a poloxamer at greater than or equal to 21 wt % of the pharmaceuticalcomposition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition to have a gelation temperature ofgreater than 10° C.

In some embodiments, the poloxamer comprises at least one of Poloxamer124, Poloxamer 188, Poloxamer 237, Poloxamer 338 or Poloxamer 407. Insome embodiments, the poloxamer comprises mixtures of two or more ofPoloxamer 124, Poloxamer 188, Poloxamer 237, Poloxamer 338 or Poloxamer407. In some embodiments, the mixture of two or more poloxamerscomprises Poloxamer 407 and Poloxamer 124. In another embodiment, thePoloxamer comprises at least one of Poloxamer 188 and Poloxamer 407 ormixtures thereof. In some embodiments, the poloxamer is Poloxamer 407.

In some embodiments, the poloxamer can be in a concentration between 21wt % and 40 wt % relative to the composition. In another embodiment, thepoloxamer is in a concentration between 21 wt % and 30 wt % relative tothe composition. In another embodiment, the poloxamer is in aconcentration between 23 wt % and 29 wt % relative to the composition.In another embodiment, the poloxamer is in a concentration between 23 wt% and 27 wt % relative to the composition. In another embodiment, thepoloxamer is in a concentration of 25 wt % relative to the composition.

In some embodiments, the gelation temperature of the pharmaceuticalcomposition is between about 11° C. and about 32° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis between is between about 15° C. and about 30° C. In some embodiments,the gelation temperature of the pharmaceutical composition is betweenabout 20° C. and about 28° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is between about 24° C.and about 26° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 15° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 20° C.In some embodiments, the gelation temperature of the pharmaceuticalcomposition is about 24° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is about 26° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis about 28° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 30° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the carboxylic acid containingcompound is a substituted or unsubstituted C₅-C₉ straight or branchedchain alkyl-CO₂H.

In any of the foregoing embodiments, the carboxylic acid containingcompound is a substituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is asubstituted C₅-C₉ straight or branched chain alkyl-CO₂H, wherein thesubstituent is —NH₂. In some examples the carboxylic acid containingcompound is an amino substituted 2-propylpentanoic acid. In someembodiments, the amino substituted 2-propylpentanoic acid is selectedfrom the group consisting of 5-amino-2-propylpentanoic acid,4-amino-2-propylpentanoic acid, 3-amino-2-propylpentanoic acid, and2-amino-2-propylpentanoic acid.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₆-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₅-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the unsubstituted C₉ branched straight chainalkyl-CO₂H carboxylic acid containing compound is Valproic acid.

In some embodiments, the Valproic acid can be in the form of an amide.

In some embodiments, the amide form of Valproic acid is the condensationproduct with an amino acid. In some embodiments, the amide of Valproicacid is selected from the group consisting of

In any of the foregoing embodiments, the amount of the carboxylic acidcontaining compound in the pharmaceutical composition is between atleast 2 wt % (weight carboxylic acid containing compound/weightpharmaceutical composition) and 20 wt %. In some embodiments, thecomposition comprises at least 4 wt % carboxylic acid. In otherembodiments, the composition comprises at least 8 wt % carboxylic acid.In some embodiments, the composition comprises at least 12 wt %carboxylic acid. In some embodiments, the composition comprises at least16 wt % carboxylic acid. In still other embodiments, the compositioncomprises at least 20 wt % carboxylic acid.

In other embodiments, the composition comprises about 4 wt % carboxylicacid. In other embodiments, the composition comprises about 8 wt %carboxylic acid. In other embodiments, the composition comprises about12 wt % carboxylic acid. In further embodiments, the compositioncomprises about 16 wt % carboxylic acid. In further embodiments, thecomposition comprises about 20 wt % carboxylic acid.

In some embodiments of the poloxamer containing pharmaceuticalcomposition, the pharmaceutically active therapeutic agent is selectedfrom the group consisting of a wnt agonist, a GSK3 inhibitor, a2-pyrimidinylaminoethylamino-2-pyridine containing compound, aN-(1H-pyrazol-4-yl)-nicotinamide containing compound, and a1,2,3,4-tetrahydro-[1,4]diazepino[6,7,]indol-7-yl)-4-(imidazo[1,2-a]pyridin-3-yl)-1H-pyrrole-2,5-dionecontaining compound.

A further aspect of the present disclosure provides a poloxamercontaining pharmaceutical composition having a gelation temperaturegreater than 10° C., the composition comprising

a) a wnt agonist or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 at greater than or equal to 21 wt % of thepharmaceutical composition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition has a gelation temperature ofgreater than 10° C.

In some embodiments, the wnt agonist is selected from the groupconsisting of

WNT Agonists CAS Number Compound 1 1084833-94-2 Compound 25 1084834-05-8BML-284 853220-52-7 IQ 1 331001-62-8 DCA 56-47-3 QS 11 944328-88-5WASP-1, ZINC00087877 352328-82-6 WAY 316606 915759-45-4 WAY-2626111123231-07-1 HLY78 854847-61-3 SKL2001 909089-13-0

In other embodiments, the wnt agonist is selected from the groupconsisting of the Compound 1, Compound 25, BML-284, QS-11, WAY 316606,WAY-262611, SKL2001, and ISX-9.

In some embodiments, Poloxamer 407 can be in a concentration between 21wt % and 40 wt % relative to the composition. In another embodiment, thePoloxamer 407 is in a concentration between 21 wt % and 30 wt % relativeto the composition. In another embodiment, the Poloxamer 407 is in aconcentration between 23 wt % and 29 wt % relative to the composition.In another embodiment, the Poloxamer 407 is in a concentration between23 wt % and 27 wt % relative to the composition. In another embodiment,the Poloxamer 407 is in a concentration of 25 wt % relative to thecomposition.

In some embodiments, the gelation temperature of the pharmaceuticalcomposition is between about 11° C. and about 32° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis between is between about 15° C. and about 30° C. In some embodiments,the gelation temperature of the pharmaceutical composition is betweenabout 20° C. and about 28° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is between about 24° C.and about 26° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 15° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 20° C.In some embodiments, the gelation temperature of the pharmaceuticalcomposition is about 24° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is about 26° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis about 28° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 30° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the carboxylic acid containingcompound is a substituted or unsubstituted C₅-C₉ straight or branchedchain alkyl-CO₂H.

In any of the foregoing embodiments, the carboxylic acid containingcompound is a substituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is asubstituted C₅-C₉ straight or branched chain alkyl-CO₂H, wherein thesubstituent is —NH₂. In some examples the carboxylic acid containingcompound is an amino substituted 2-propylpentanoic acid. In someembodiments, the amino substituted 2-propylpentanoic acid is selectedfrom the group consisting of 5-amino-2-propylpentanoic acid,4-amino-2-propylpentanoic acid, 3-amino-2-propylpentanoic acid, and2-amino-2-propylpentanoic acid.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₆-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₅-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the unsubstituted C₉ branched straight chainalkyl-CO₂H carboxylic acid containing compound is Valproic acid.

In some embodiments, the Valproic acid can be in the form of an amide.In some embodiments, the amide form of Valproic acid is the condensationproduct with an amino acid.

In some embodiments, the amide of Valproic acid is selected from thegroup consisting of

In any of the foregoing embodiments, the amount of the carboxylic acidcontaining compound in the pharmaceutical composition is between atleast 2 wt % (weight carboxylic acid containing compound/weightpharmaceutical composition) and 20 wt %. In some embodiments, thecomposition comprises at least 4 wt % carboxylic acid. In otherembodiments, the composition comprises at least 8 wt % carboxylic acid.In some embodiments, the composition comprises at least 12 wt %carboxylic acid. In some embodiments, the composition comprises at least16 wt % carboxylic acid. In still other embodiments, the compositioncomprises at least 20 wt % carboxylic acid.

In other embodiments, the composition comprises about 4 wt % carboxylicacid. In other embodiments, the composition comprises about 8 wt %carboxylic acid. In other embodiments, the composition comprises about12 wt % carboxylic acid. In further embodiments, the compositioncomprises about 16 wt % carboxylic acid. In further embodiments, thecomposition comprises about 20 wt % carboxylic acid.

A further aspect of the present disclosure provides a poloxamercontaining pharmaceutical composition having a gelation temperaturegreater than 10° C., the composition comprising:

a) a GSK3 inhibitor or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 at greater than or equal to 21 wt % of thepharmaceutical composition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition has a gelation temperature ofgreater than 10° C.

In some embodiments, the pharmaceutically active therapeutic agent is aGSK3 inhibitor. In some embodiments, the GSK3 inhibitor is a GSK3 alphainhibitor. In some embodiments, the GSK alpha inhibitor is selected fromthe group consisting of

GSKalpha inhibitor CAS Number GSK-3 inhibitor XXII 1195901-31-5 AT 7519844442-38-2 Compound 4a 1627557-91-8 Compound 4t 1627558-10-4 Compound4z 1627558-16-0 Compound 14 583038-63-5 Compound 23 583038-76-0Pyrazolopyridine 34 583039-27-4 Compound 18 405223-20-3 Compound 19405223-71-4 Compound 15b 1374671-66-5 Compound 14d 1374671-64-3 Compound27 1820758-44-8 AZD1080 612487-72-6 Compound 39 1772824-10-8 Compound 291772823-37-6 Compound 33 1772823-64-9 Tivantinib 905854-02-6 I5264217-24-5 Compound 90 91322-11-1 Compound 92 1043429-30-6 CompoundΛ-OS1 1291104-51-2 1292843-11-8 Compound 3 1498285-39-4 1498285-48-5Compound (R)-DW12 1047684-07-0 BRD4003 chiral 1597439-60-5 BRD4003chiral 1597439-59-2 Compound 8 1597439-01-4 Compound 9 1597439-02-52056261-29-9 Compound 11 1597439-12-7 BRD1172 1597438-86-2 Compound 161597440-17-9 BRD1652 1597438-93-1 AR-A014418 487021-52-3 CREB knockdown

In some embodiments, the GSK3-alpha inhibitors are selected from thegroup consisting of GSK-alpha Inhibitor XXII, Compound 4a, Compound 4t,Compound 4z, Compound 14, Compound 23, Compound 18, Compound 19,Compound 15b, Compound 14d, AZD-1080, Compound 39, Compound 29, Compound33, Compound 92, and Compound A-OS1

In some embodiments, the pharmaceutically active therapeutic agent is aGSK3 inhibitor is a GSK3 beta inhibitor.

In some embodiments, the GSK3 Inhibitor is selected from the groupconsisting of:

GSK3β Inhibitor CAS Reg. No. Valproic Acid, Sodium Salt 99-66-1 Bikinin188011-69-0 Hymenialdisine 82005-12-7 Aloisine A 496864-16-5 Aloisine B496864-14-3 TWS119 1507095-58-0 CT20026 403808-63-9 CHIR99021 (CT99021)252917-06-9 CHIR98014 (CT98014) 252935-94-7 CHIR98023 (CT98023)252904-84-0 CHIR98024 (CT98024) 556813-39-9 GSK-3β Inhibitor XVIII1139875-74-3 CGP60474 164658-13-3 AZD2858 (AR28) 486424-20-8 CID 755673521937-07-5 TCS 2002 1005201-24-0 Dibromocantharelline 101481-34-9 ML3201597438-84-0 Flavopiridol 146426-40-6 Compound 100 744255-19-4 Hymenidin107019-95-4 6-Bromoindirubin-3-acetoxime 667463-85-6 GSK-3 Inhibitor IX667463-62-9 Indirubin-3′-monoxime 160807-49-85-Iodo-indirubin-3′-monoxime 331467-03-9 Indirubin-5-sulfonic acidsodium salt 331467-05-1 Indirubin 479-41-4 GSK-3 Inhibitor X 740841-15-0Lithium Chloride Beryllium Zinc Tungstate Compound 39 1772824-10-8Compound 29 1772823-37-6 Compound 33 1772823-64-9 Compound 29436866-61-4 Compound 46 682807-74-5 Compound 5a 436866-54-5 GF109203x176504-36-2 Ro318220 125314-64-9 Bisindolylmaleimide X HCl 131848-97-0Enzastaurin (LY317615) 170364-57-5 I5 264217-24-5 SB-216763 280744-09-4SB-415286 (SB-41528) 264218-23-7 3F8 159109-11-2 TCS 21311 1260181-14-3GSK-3 inhibitor 1 603272-51-1 LY2090314 603288-22-8 603281-31-8603281-31-8 IM-12 1129669-05-1 Compound 34 396091-16-0 KT 5720108068-98-0 Isogranulatimide 244148-46-7 GSK-3β Inhibitor XI 626604-39-5BIP-135 941575-71-9 CP21R7 125314-13-8 Tivantinib 905854-02-6 CompoundΛ-OS1 1291104-51-2, 1292843-11-8 HB12 800384-87-6 DW12 861251-33-4 NP309937810-13-4 (RRu)-HB1229 (RRu)-NP549 Compound 3 1498285-39-4,1498285-48-5 Compound (R)-DW12 1047684-07-0 Staurosporine 62996-74-1GSK-3beta Inhibitor XXVI 871843-09-3 Manzamine A 104196-68-1 TC-G 241257256-44-2 Compound 14d 1374671-64-3 Compound 15b 1374671-66-5Compound 20x 1005201-80-8 GSK-3 Inhibitor II 478482-75-6 GSK3 Inhibitor,2 1377154-01-2 SU9516 77090-84-1 AZD-1080 612487-72-6 Kenpaullone142273-20-9 Compound 17b 408532-42-3 Azakenpaullone 676596-65-9Alsterpaullone 237430-03-4 Alsterpaullone CN Ethyl 852529-97-0Cazpaullone 914088-64-5 FRATtide L803 L803-mts GSK-3 Inhibitor XXII1195901-31-5 Compound 4a 1627557-91-8 Compound 4t 1627558-10-4 Compound4z 1627558-16-0 AT 7519 844442-38-2 Pyrazolopyridine 9 923029-74-7Pyrazolopyridine 18 405221-39-8 Pyrazolopyridine 34 583039-27-4 Compound14 583038-63-5 Compound 23 583038-76-0 Compound 14 583038-63-5 Compound18 405223-20-3 Compound 19 405223-71-4 NSC 693868 (Compound 1)40254-90-8 Compound 150 1282042-18-5 GSK-3 Inhibitor XIII 404828-08-6VP0.7 331963-23-6 1132813-46-7 1132812-98-6 950727-66-9 NSC 693868(Compound 1) 40254-90-8 Compound 17 62673-69-2 GSK-3β Inhibitor VII99-73-0 GSK-3β Inhibitor VI 62673-69-2 Palinurin 254901-27-4 Tricantin853885-55-9 GSK-3β Inhibitor I 327036-89-5 NP031115 1400575-57-6NP031112 (Tideglusib) 865854-05-3 Compound 90 91322-11-1 Compound 921043429-30-6 GSK-3β Inh. VIII AR-A014418 487021-52-3 A-10707221384424-80-9 NP-103 No Structure CG-301338 No Structure SAR 502250 NoStructure XD-4241 No Structure CEP-16805 No Structure AZ13282107 NoStructure SAR 502250 (Sanofi) 1073653-58-3 Compound 27 2025388-25-2Compound 12 2025388-10-5

In certain embodiments, the GSK3β Inhibitor are selected from the groupconsisting of Valproic Acid Sodium Salt, CT20026, CHIR99021 (CT99021),CHIR98014 (CT98014), CHIR98023 (CT98023), CHIR98024 (CT98024), TCS 2002,Compound 39, Compound 29, Compound 33, TCS 21311, LY2090314,603281-31-8, Compound 34, Compound 14d, Compound 15b, Compound 20x,AZD-1080, Kenpaullone, Cazpaullone, GSK-3 Inhibitor XXII, Compound 4a,Compound 4t, Compound 4z, Pyrazolopyridine 9, Compound 14, Compound 23,Compound 14, Compound 18, and Compound 19.

In certain embodiments, the GSK3P Inhibitor are selected from the groupconsisting of Valproic Acid Sodium Salt, CHIR99021 (CT99021), CHIR98014(CT98014), CHIR98023 (CT98023), CHIR98024 (CT98024), Compound 39,Compound 29, LY2090314, 603281-31-8, Compound 34, Compound 14d, Compound15b, Compound 20x, AZD-1080, Cazpaullone, GSK-3 Inhibitor XXII, Compound4t, Compound 4z, Pyrazolopyridine 9, Compound 14, Compound 23, Compound14, Compound 18, and Compound 19.

In some embodiments, the Poloxamer 407 can be in a concentration between21 wt % and 40 wt % relative to the composition. In another embodiment,the Poloxamer 407 is in a concentration between 21 wt % and 30 wt %relative to the composition. In another embodiment, the Poloxamer 407 isin a concentration between 23 wt % and 29 wt % relative to thecomposition. In another embodiment, the Poloxamer 407 is in aconcentration between 23 wt % and 27 wt % relative to the composition.In another embodiment, the Poloxamer 407 is in a concentration of 25 wt% relative to the composition.

In some embodiments, the gelation temperature of the pharmaceuticalcomposition is between about 11° C. and about 32° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis between is between about 15° C. and about 30° C. In some embodiments,the gelation temperature of the pharmaceutical composition is betweenabout 20° C. and about 28° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is between about 24° C.and about 26° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 15° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 20° C.In some embodiments, the gelation temperature of the pharmaceuticalcomposition is about 24° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is about 26° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis about 28° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 30° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the carboxylic acid containingcompound is a substituted or unsubstituted C₅-C₉ straight or branchedchain alkyl-CO₂H.

In any of the foregoing embodiments, the carboxylic acid containingcompound is a substituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is asubstituted C₅-C₉ straight or branched chain alkyl-CO₂H, wherein thesubstituent is —NH₂. In some examples the carboxylic acid containingcompound is an amino substituted 2-propylpentanoic acid. In someembodiments, the amino substituted 2-propylpentanoic acid is selectedfrom the group consisting of 5-amino-2-propylpentanoic acid,4-amino-2-propylpentanoic acid, 3-amino-2-propylpentanoic acid, and2-amino-2-propylpentanoic acid.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₆-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₈-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the unsubstituted C₉ branched straight chainalkyl-CO₂H carboxylic acid containing compound is Valproic acid.

In some embodiments, the Valproic acid can be in the form of an amide.In some embodiments, the amide form of Valproic acid is the condensationproduct with an amino acid. In some embodiments, the amide of Valproicacid is selected from the group consisting of

In any of the foregoing embodiments, the amount of the carboxylic acidcontaining compound in the pharmaceutical composition is between atleast 2 wt % (weight carboxylic acid containing compound/weightpharmaceutical composition) and 20 wt %. In some embodiments, thecomposition comprises at least 4 wt % carboxylic acid. In otherembodiments, the composition comprises at least 8 wt % carboxylic acid.In some embodiments, the composition comprises at least 12 wt %carboxylic acid. In some embodiments, the composition comprises at least16 wt % carboxylic acid. In still other embodiments, the compositioncomprises at least 20 wt % carboxylic acid.

In other embodiments, the composition comprises about 4 wt % carboxylicacid. In other embodiments, the composition comprises about 8 wt %carboxylic acid. In other embodiments, the composition comprises about12 wt % carboxylic acid. In further embodiments, the compositioncomprises about 16 wt % carboxylic acid. In further embodiments, thecomposition comprises about 20 wt % carboxylic acid.

A further aspect of the present disclosure provides a poloxamercontaining pharmaceutical composition having a gelation temperaturegreater than 10° C., the composition comprising:

a) a 2-pyrimidinylaminoethylamino-2-pyridine compound;

b) Poloxamer 407 at greater than or equal to 21 wt % of thepharmaceutical composition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition has a gelation temperature ofgreater than 10° C.

In some embodiments, the 2-pyrimidinylaminoethylamino-2-pyridinecompound is a2,4-dichlorophenyl-5-(1H-imidazol-2-yl)-2-pyrimidinylaminoethylamino-3-pyridinecompound. In some embodiments, the pyrimidine containing compound is6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrilewhich has the Formula I and pharmaceutically acceptable salts thereof:

The compound of Formula I is also known as CHIR99021.

In some embodiments, the Poloxamer 407 can be in a concentration between21 wt % and 40 wt % relative to the composition. In another embodiment,the Poloxamer 407 is in a concentration between 21 wt % and 30 wt %relative to the composition. In another embodiment, the Poloxamer 407 isin a concentration between 23 wt % and 29 wt % relative to thecomposition. In another embodiment, the Poloxamer 407 is in aconcentration between 23 wt % and 27 wt % relative to the composition.In another embodiment, the Poloxamer 407 is in a concentration of 25 wt% relative to the composition.

In some embodiments, the gelation temperature of the pharmaceuticalcomposition is between about 11° C. and about 32° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis between is between about 15° C. and about 30° C. In some embodiments,the gelation temperature of the pharmaceutical composition is betweenabout 20° C. and about 28° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is between about 24° C.and about 26° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 15° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 20° C.In some embodiments, the gelation temperature of the pharmaceuticalcomposition is about 24° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is about 26° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis about 28° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 30° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the carboxylic acid containingcompound is a substituted or unsubstituted C₅-C₉ straight or branchedchain alkyl-CO₂H.

In any of the foregoing embodiments, the carboxylic acid containingcompound is a substituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is asubstituted C₅-C₉ straight or branched chain alkyl-CO₂H, wherein thesubstituent is —NH₂. In some examples the carboxylic acid containingcompound is an amino substituted 2-propylpentanoic acid. In someembodiments, the amino substituted 2-propylpentanoic acid is selectedfrom the group consisting of 5-amino-2-propylpentanoic acid,4-amino-2-propylpentanoic acid, 3-amino-2-propylpentanoic acid, and2-amino-2-propylpentanoic acid.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₆-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₈-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the unsubstituted C₉ branched straight chainalkyl-CO₂H carboxylic acid containing compound is Valproic acid.

In some embodiments, the Valproic acid can be in the form of an amide.In some embodiments, the amide form of Valproic acid is the condensationproduct with an amino acid. In some embodiments, the amide of Valproicacid is selected from the group consisting of

In any of the foregoing embodiments, the amount of the carboxylic acidcontaining compound in the pharmaceutical composition is between atleast 2 wt % (weight carboxylic acid containing compound/weightpharmaceutical composition) and 20 wt %. In some embodiments, thecomposition comprises at least 4 wt % carboxylic acid. In otherembodiments, the composition comprises at least 8 wt % carboxylic acid.In some embodiments, the composition comprises at least 12 wt %carboxylic acid. In some embodiments, the composition comprises at least16 wt % carboxylic acid. In still other embodiments, the compositioncomprises at least 20 wt % carboxylic acid.

In other embodiments, the composition comprises about 4 wt % carboxylicacid. In other embodiments, the composition comprises about 8 wt %carboxylic acid. In other embodiments, the composition comprises about12 wt % carboxylic acid. In further embodiments, the compositioncomprises about 16 wt % carboxylic acid. In further embodiments, thecomposition comprises about 20 wt % carboxylic acid.

A further aspect of the present disclosure provides poloxamer containingpharmaceutical composition having a gelation temperature greater than10° C., the composition comprising:

a) an N-(alkylcarbamoyl)-1H-pyrazol-4-yl)-nicotinamide containingcompound or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 at greater than or equal to 21 wt % of thepharmaceutical composition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition has a gelation temperature ofgreater than 10° C.

In some embodiments, the N-(1H-pyrazol-4-yl)-nicotinamide containingcompound is an N-(alkylcarbamoyl)-1H-pyrazol-4-yl)-nicotinamidecontaining compound. In some embodiments, theN-(1H-pyrazol-4-yl)-nicotinamide containing compound isN-(3-((3-isopropoxypropyl)carbamoyl)-1H-pyrazol-4-yl)-6-methylnicotinamidewhich has the Formula II and pharmaceutically acceptable salts thereof:

The compound of Formula II is also known as GSK-3 Inhibitor XXII.

In some embodiments, the Poloxamer 407 can be in a concentration between21 wt % and 40 wt % relative to the composition. In another embodiment,the Poloxamer 407 is in a concentration between 21 wt % and 30 wt %relative to the composition. In another embodiment, the Poloxamer 407 isin a concentration between 23 wt % and 29 wt % relative to thecomposition. In another embodiment, the Poloxamer 407 is in aconcentration between 23 wt % and 27 wt % relative to the composition.In another embodiment, the Poloxamer 407 is in a concentration of 25 wt% relative to the composition.

In some embodiments, the gelation temperature of the pharmaceuticalcomposition is between about 11° C. and about 32° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis between is between about 15° C. and about 30° C. In some embodiments,the gelation temperature of the pharmaceutical composition is betweenabout 20° C. and about 28° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is between about 24° C.and about 26° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 15° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 20° C.In some embodiments, the gelation temperature of the pharmaceuticalcomposition is about 24° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is about 26° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis about 28° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 30° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the carboxylic acid containingcompound is a substituted or unsubstituted C₅-C₉ straight or branchedchain alkyl-CO₂H.

In any of the foregoing embodiments, the carboxylic acid containingcompound is a substituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is asubstituted C₅-C₉ straight or branched chain alkyl-CO₂H, wherein thesubstituent is —NH₂. In some examples the carboxylic acid containingcompound is an amino substituted 2-propylpentanoic acid. In someembodiments, the amino substituted 2-propylpentanoic acid is selectedfrom the group consisting of 5-amino-2-propylpentanoic acid,4-amino-2-propylpentanoic acid, 3-amino-2-propylpentanoic acid, and2-amino-2-propylpentanoic acid.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₆-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₅-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the unsubstituted C₉ branched straight chainalkyl-CO₂H carboxylic acid containing compound is Valproic acid.

In some embodiments, the Valproic acid can be in the form of an amide.In some embodiments, the amide form of Valproic acid is the condensationproduct with an amino acid.

In some embodiments, the amide of Valproic acid is selected from thegroup consisting of

In any of the foregoing embodiments, the amount of the carboxylic acidcontaining compound in the pharmaceutical composition is between atleast 2 wt % (weight carboxylic acid containing compound/weightpharmaceutical composition) and 20 wt %. In some embodiments, thecomposition comprises at least 4 wt % carboxylic acid. In otherembodiments, the composition comprises at least 8 wt % carboxylic acid.In some embodiments, the composition comprises at least 12 wt %carboxylic acid. In some embodiments, the composition comprises at least16 wt % carboxylic acid. In still other embodiments, the compositioncomprises at least 20 wt % carboxylic acid.

In other embodiments, the composition comprises about 4 wt % carboxylicacid. In other embodiments, the composition comprises about 8 wt %carboxylic acid. In other embodiments, the composition comprises about12 wt % carboxylic acid. In further embodiments, the compositioncomprises about 16 wt % carboxylic acid. In further embodiments, thecomposition comprises about 20 wt % carboxylic acid.

A further aspect of the present disclosure provides poloxamer containingpharmaceutical composition having a gelation temperature greater than10° C., the composition comprising:

a) a 1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indole containingcompound;

b) Poloxamer 407 at greater than or equal to 21 wt % of thepharmaceutical composition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition has a gelation temperature ofgreater than 10° C.

In some embodiments, the1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indole containing compound isa3-(1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-1H-pyrrole-2,5-dionecontaining compound. In some embodiments, the1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indole containing compound isa3-(imidazo[1,2-a]pyridin-3-yl)-4-(1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-1H-pyrrole-2,5-dionecontaining compound. In some embodiments,1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indole containing compound is3-(9-fluoro-2-(piperidine-1-carbonyl)-1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-4-(imidazo[1,2-a]pyridin-3-yl)-1H-pyrrole-2,5-dionewhich has the Formula III and pharmaceutically acceptable salts thereof:

The compound of Formula III is also known as LY2090314.

In some embodiments, the Poloxamer 407 can be in a concentration between21 wt % and 40 wt % relative to the composition. In another embodiment,the Poloxamer 407 is in a concentration between 21 wt % and 30 wt %relative to the composition. In another embodiment, the Poloxamer 407 isin a concentration between 23 wt % and 29 wt % relative to thecomposition. In another embodiment, the Poloxamer 407 is in aconcentration between 23 wt % and 27 wt % relative to the composition.In another embodiment, the Poloxamer 407 is in a concentration of 25 wt% relative to the composition.

In some embodiments, the gelation temperature of the pharmaceuticalcomposition is between about 11° C. and about 32° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis between is between about 15° C. and about 30° C. In some embodiments,the gelation temperature of the pharmaceutical composition is betweenabout 20° C. and about 28° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is between about 24° C.and about 26° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 15° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 20° C.In some embodiments, the gelation temperature of the pharmaceuticalcomposition is about 24° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is about 26° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis about 28° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 30° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the carboxylic acid containingcompound is a substituted or unsubstituted C₅-C₉ straight or branchedchain alkyl-CO₂H.

In any of the foregoing embodiments, the carboxylic acid containingcompound is a substituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is asubstituted C₅-C₉ straight or branched chain alkyl-CO₂H, wherein thesubstituent is —NH₂. In some examples the carboxylic acid containingcompound is an amino substituted 2-propylpentanoic acid. In someembodiments, the amino substituted 2-propylpentanoic acid is selectedfrom the group consisting of 5-amino-2-propylpentanoic acid,4-amino-2-propylpentanoic acid, 3-amino-2-propylpentanoic acid, and2-amino-2-propylpentanoic acid.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₆-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₈-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is anunsubstituted C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the unsubstituted C₉ branched straight chainalkyl-CO₂H carboxylic acid containing compound is Valproic acid.

In some embodiments, the Valproic acid can be in the form of an amide.In some embodiments, the amide form of Valproic acid is the condensationproduct with an amino acid. In some embodiments, the amide of Valproicacid is selected from the group consisting of

In any of the foregoing embodiments, the amount of the carboxylic acidcontaining compound in the pharmaceutical composition is between atleast 2 wt % (weight carboxylic acid containing compound/weightpharmaceutical composition) and 20 wt %. In some embodiments, thecomposition comprises at least 4 wt % carboxylic acid. In otherembodiments, the composition comprises at least 8 wt % carboxylic acid.In some embodiments, the composition comprises at least 12 wt %carboxylic acid. In some embodiments, the composition comprises at least16 wt % carboxylic acid. In still other embodiments, the compositioncomprises at least 20 wt % carboxylic acid.

In other embodiments, the composition comprises about 4 wt % carboxylicacid. In other embodiments, the composition comprises about 8 wt %carboxylic acid. In other embodiments, the composition comprises about12 wt % carboxylic acid. In further embodiments, the compositioncomprises about 16 wt % carboxylic acid. In further embodiments, thecomposition comprises about 20 wt % carboxylic acid.

A further aspect of the present disclosure provides a poloxamercontaining pharmaceutical composition having a gelation temperaturegreater than 10° C., the composition comprising:

a) a wnt agonist or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 at greater than or equal to 21 wt % of thepharmaceutical composition; and

c) Valproic acid;

wherein the pharmaceutical composition has a gelation temperature ofgreater than 10° C.

In some embodiments, the Poloxamer 407 can be in a concentration between21 wt % and 40 wt % relative to the composition. In another embodiment,the Poloxamer 407 is in a concentration between 21 wt % and 30 wt %relative to the composition. In another embodiment, the Poloxamer 407 isin a concentration between 23 wt % and 29 wt % relative to thecomposition. In another embodiment, the Poloxamer 407 is in aconcentration between 23 wt % and 27 wt % relative to the composition.In another embodiment, the Poloxamer 407 is in a concentration of 25 wt% relative to the composition.

In some embodiments, the gelation temperature of the pharmaceuticalcomposition is between about 11° C. and about 32° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis between is between about 15° C. and about 30° C. In some embodiments,the gelation temperature of the pharmaceutical composition is betweenabout 20° C. and about 28° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is between about 24° C.and about 26° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 15° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 20° C.In some embodiments, the gelation temperature of the pharmaceuticalcomposition is about 24° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is about 26° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis about 28° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 30° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the amount of the Valproic acid inthe pharmaceutical composition is between least 2 wt % (weight Valproicacid/weight pharmaceutical composition) and 20 wt %. In someembodiments, the composition comprises at least 4 wt % Valproic acid. Inother embodiments, the composition comprises at least 8 wt % Valproicacid. In some embodiments, the composition comprises at least 12 wt %Valproic acid. In some embodiments, the composition comprises at least16 wt % Valproic acid. In still other embodiments, the compositioncomprises at least 20 wt % Valproic acid.

In other embodiments, the composition comprises about 4 wt % Valproicacid. In other embodiments, the composition comprises about 8 wt %Valproic acid. In other embodiments, the composition comprises about 12wt % Valproic acid. In further embodiments, the composition comprisesabout 16 wt % Valproic acid. In further embodiments, the compositioncomprises about 20 wt % Valproic acid.

A further aspect of the present disclosure provides a poloxamercontaining pharmaceutical composition having a gelation temperaturegreater than 10° C., the composition comprising:

a) a GSK3 inhibitor or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 at greater than or equal to 21 wt % of thepharmaceutical composition; and

c) Valproic acid;

wherein the pharmaceutical composition has a gelation temperature ofgreater than 10° C.

In some embodiments, the Poloxamer 407 can be in a concentration between21 wt % and 40 wt % relative to the composition. In another embodiment,the Poloxamer 407 is in a concentration between 21 wt % and 30 wt %relative to the composition. In another embodiment, the Poloxamer 407 isin a concentration between 23 wt % and 29 wt % relative to thecomposition. In another embodiment, the Poloxamer 407 is in aconcentration between 23 wt % and 27 wt % relative to the composition.In another embodiment, the Poloxamer 407 is in a concentration of 25 wt% relative to the composition.

In some embodiments, the gelation temperature of the pharmaceuticalcomposition is between about 11° C. and about 32° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis between is between about 15° C. and about 30° C. In some embodiments,the gelation temperature of the pharmaceutical composition is betweenabout 20° C. and about 28° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is between about 24° C.and about 26° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 15° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 20° C.In some embodiments, the gelation temperature of the pharmaceuticalcomposition is about 24° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is about 26° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis about 28° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 30° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the amount of the Valproic acid inthe pharmaceutical composition is between least 2 wt % (weight Valproicacid/weight pharmaceutical composition) and 20 wt %. In someembodiments, the composition comprises at least 4 wt % Valproic acid. Inother embodiments, the composition comprises at least 8 wt % Valproicacid. In some embodiments, the composition comprises at least 12 wt %Valproic acid. In some embodiments, the composition comprises at least16 wt % Valproic acid. In still other embodiments, the compositioncomprises at least 20 wt % Valproic acid.

In other embodiments, the composition comprises about 4 wt % Valproicacid. In other embodiments, the composition comprises about 8 wt %Valproic acid. In other embodiments, the composition comprises about 12wt % Valproic acid. In further embodiments, the composition comprisesabout 16 wt % Valproic acid. In further embodiments, the compositioncomprises about 20 wt % Valproic acid.

A further aspect of the present disclosure provides poloxamer containingpharmaceutical composition having a gelation temperature greater than10° C., the composition comprising:

a) a 2-pyrimidinylaminoethylamino-2-pyridine compound;

b) Poloxamer 407 at greater than or equal to 21 wt % of thepharmaceutical composition; and

c) Valproic acid;

wherein the pharmaceutical composition has a gelation temperature ofgreater than 10° C.

In some embodiments, the 2-pyrimidinylaminoethylamino-2-pyridinecompound is a2,4-dichlorophenyl-5-(1H-imidazol-2-yl)-2-pyrimidinylaminoethylamino-2-pyridinecompound. In some embodiments, the pyrimidine containing compound is6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrilewhich has the Formula I and pharmaceutically acceptable salts thereof:

In some embodiments, the Poloxamer 407 can be in a concentration between21 wt % and 40 wt % relative to the composition. In another embodiment,the Poloxamer 407 is in a concentration between 21 wt % and 30 wt %relative to the composition. In another embodiment, the Poloxamer 407 isin a concentration between 23 wt % and 29 wt % relative to thecomposition. In another embodiment, the Poloxamer 407 is in aconcentration between 23 wt % and 27 wt % relative to the composition.In another embodiment, the Poloxamer 407 is in a concentration of 25 wt% relative to the composition.

In some embodiments, the gelation temperature of the pharmaceuticalcomposition is between about 11° C. and about 32° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis between is between about 15° C. and about 30° C. In some embodiments,the gelation temperature of the pharmaceutical composition is betweenabout 20° C. and about 28° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is between about 24° C.and about 26° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 15° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 20° C.In some embodiments, the gelation temperature of the pharmaceuticalcomposition is about 24° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is about 26° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis about 28° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 30° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the amount of the Valproic acid inthe pharmaceutical composition is between least 2 wt % (weight Valproicacid/weight pharmaceutical composition) and 20 wt %. In someembodiments, the composition comprises at least 4 wt % Valproic acid. Inother embodiments, the composition comprises at least 8 wt % Valproicacid. In some embodiments, the composition comprises at least 12 wt %Valproic acid. In some embodiments, the composition comprises at least16 wt % Valproic acid. In still other embodiments, the compositioncomprises at least 20 wt % Valproic acid.

In other embodiments, the composition comprises about 4 wt % Valproicacid. In other embodiments, the composition comprises about 8 wt %Valproic acid. In other embodiments, the composition comprises about 12wt % Valproic acid. In further embodiments, the composition comprisesabout 16 wt % Valproic acid. In further embodiments, the compositioncomprises about 20 wt % Valproic acid.

A further aspect of the present disclosure provides poloxamer containingpharmaceutical composition having a gelation temperature greater than10° C., the composition comprising:

a) an N-(1H-pyrazol-4-yl)-nicotinamide containing compound or apharmaceutically acceptable salt thereof;

b) Poloxamer 407 at greater than or equal to 21 wt % of thepharmaceutical composition; and

c) Valproic acid;

wherein the pharmaceutical composition has a gelation temperature ofgreater than 10° C.

In some embodiments, the N-(1H-pyrazol-4-yl)-nicotinamide containingcompound is an N-(alkylcarbamoyl)-1H-pyrazol-4-yl)-nicotinamidecontaining compound.

In some embodiments, the N-(1H-pyrazol-4-yl)-nicotinamide containingcompound isN-(3-((3-isopropoxypropyl)carbamoyl)-1H-pyrazol-4-yl)-6-methylnicotinamidewhich has the structure in Formula II and pharmaceutically acceptablesalts thereof:

In some embodiments, the Poloxamer 407 can be in a concentration between21 wt % and 40 wt % relative to the composition. In another embodiment,the Poloxamer 407 is in a concentration between 21 wt % and 30 wt %relative to the composition. In another embodiment, the Poloxamer 407 isin a concentration between 23 wt % and 29 wt % relative to thecomposition. In another embodiment, the Poloxamer 407 is in aconcentration between 23 wt % and 27 wt % relative to the composition.In another embodiment, the Poloxamer 407 is in a concentration of 25 wt% relative to the composition.

In some embodiments, the gelation temperature of the pharmaceuticalcomposition is between about 11° C. and about 32° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis between is between about 15° C. and about 30° C. In some embodiments,the gelation temperature of the pharmaceutical composition is betweenabout 20° C. and about 28° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is between about 24° C.and about 26° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 15° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 20° C.In some embodiments, the gelation temperature of the pharmaceuticalcomposition is about 24° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is about 26° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis about 28° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 30° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the amount of the Valproic acid inthe pharmaceutical composition is between least 2 wt % (weight Valproicacid/weight pharmaceutical composition) and 20 wt %. In someembodiments, the composition comprises at least 4 wt % Valproic acid. Inother embodiments, the composition comprises at least 8 wt % Valproicacid. In some embodiments, the composition comprises at least 12 wt %Valproic acid. In some embodiments, the composition comprises at least16 wt % Valproic acid. In still other embodiments, the compositioncomprises at least 20 wt % Valproic acid.

In other embodiments, the composition comprises about 4 wt % Valproicacid. In other embodiments, the composition comprises about 8 wt %Valproic acid. In other embodiments, the composition comprises about 12wt % Valproic acid. In further embodiments, the composition comprisesabout 16 wt % Valproic acid. In further embodiments, the compositioncomprises about 20 wt % Valproic acid.

A further aspect of the present disclosure provides a poloxamercontaining pharmaceutical composition having a gelation temperaturegreater than 10° C., the composition comprising:

a) a 1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indolyl) containingcompound containing compound or a pharmaceutically acceptable saltthereof;

b) Poloxamer 407 at greater than or equal to 21 wt % of thepharmaceutical composition; and

c) Valproic acid;

wherein the pharmaceutical composition has a gelation temperature ofgreater than 10° C.

In some embodiments, the1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indolyl) containing compoundis a1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indole-7-yl)-1H-pyrrole-2,5-dionecontaining compound. In some embodiments, the1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indolyl) containing compoundis a1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indole-7-yl)-4-(imidazo[1,2-a]pyridin-3-yl)-1H-pyrrole-2,5-dionecontaining compound. In some embodiments, the1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indolyl) containing compoundis3-(9-fluoro-2-(piperidine-1-carbonyl)-1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indole-7-yl)-4-(imidazo[1,2-a]pyridin-3-yl)-1H-pyrrole-2,5-dionewhich has the Formula III and pharmaceutically acceptable salts thereof:

In some embodiments, the Poloxamer 407 can be in a concentration between21 wt % and 40 wt % relative to the composition. In another embodiment,the Poloxamer 407 is in a concentration between 21 wt % and 30 wt %relative to the composition. In another embodiment, the Poloxamer 407 isin a concentration between 23 wt % and 29 wt % relative to thecomposition. In another embodiment, the Poloxamer 407 is in aconcentration between 23 wt % and 27 wt % relative to the composition.In another embodiment, the Poloxamer 407 is in a concentration of 25 wt% relative to the composition.

In some embodiments, the gelation temperature of the pharmaceuticalcomposition is between about 11° C. and about 32° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis between is between about 15° C. and about 30° C. In some embodiments,the gelation temperature of the pharmaceutical composition is betweenabout 20° C. and about 28° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is between about 24° C.and about 26° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 15° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 20° C.In some embodiments, the gelation temperature of the pharmaceuticalcomposition is about 24° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is about 26° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis about 28° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 30° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the amount of the Valproic acid inthe pharmaceutical composition is between least 2 wt % (weight Valproicacid/weight pharmaceutical composition) and 20 wt %. In someembodiments, the composition comprises at least 4 wt % Valproic acid. Inother embodiments, the composition comprises at least 8 wt % Valproicacid. In some embodiments, the composition comprises at least 12 wt %Valproic acid. In some embodiments, the composition comprises at least16 wt % Valproic acid. In still other embodiments, the compositioncomprises at least 20 wt % Valproic acid.

In other embodiments, the composition comprises about 4 wt % Valproicacid. In other embodiments, the composition comprises about 8 wt %Valproic acid. In other embodiments, the composition comprises about 12wt % Valproic acid. In further embodiments, the composition comprisesabout 16 wt % Valproic acid. In further embodiments, the compositioncomprises about 20 wt % Valproic acid.

A further aspect of the present disclosure provides a poloxamercontaining pharmaceutical composition having a gelation temperaturegreater than 10° C., the composition comprising

a) a compound of the Formula I

or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 in weight percentage of more than 21% wt; and

c) Valproic acid;

wherein the pharmaceutical composition has a gelation temperature ofgreater than 10° C.

In some embodiments, the Poloxamer 407 can be in a concentration between21 wt % and 40 wt % relative to the composition. In another embodiment,the Poloxamer 407 is in a concentration between 21 wt % and 30 wt %relative to the composition. In another embodiment, the Poloxamer 407 isin a concentration between 23 wt % and 29 wt % relative to thecomposition. In another embodiment, the Poloxamer 407 is in aconcentration between 23 wt % and 27 wt % relative to the composition.In another embodiment, the Poloxamer 407 is in a concentration of 25 wt% relative to the composition.

In some embodiments, the gelation temperature of the pharmaceuticalcomposition is between about 11° C. and about 32° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis between is between about 15° C. and about 30° C. In some embodiments,the gelation temperature of the pharmaceutical composition is betweenabout 20° C. and about 28° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is between about 24° C.and about 26° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 15° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 20° C.In some embodiments, the gelation temperature of the pharmaceuticalcomposition is about 24° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is about 26° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis about 28° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 30° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the amount of the Valproic acid inthe pharmaceutical composition is between least 2 wt % (weight Valproicacid/weight pharmaceutical composition) and 20 wt %. In someembodiments, the composition comprises at least 4 wt % Valproic acid. Inother embodiments, the composition comprises at least 8 wt % Valproicacid. In some embodiments, the composition comprises at least 12 wt %Valproic acid. In some embodiments, the composition comprises at least16 wt % Valproic acid. In still other embodiments, the compositioncomprises at least 20 wt % Valproic acid.

In other embodiments, the composition comprises about 4 wt % Valproicacid. In other embodiments, the composition comprises about 8 wt %Valproic acid. In other embodiments, the composition comprises about 12wt % Valproic acid. In further embodiments, the composition comprisesabout 16 wt % Valproic acid. In further embodiments, the compositioncomprises about 20 wt % Valproic acid.

A further aspect of the present disclosure provides a poloxamercontaining pharmaceutical composition having a gelation temperaturegreater than 10° C., the composition comprising:

a) a compound of Formula II

or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 in weight percentage of more than 21% wt; and

c) Valproic acid;

wherein the pharmaceutical composition has a gelation temperature ofgreater than 10° C.

In some embodiments, the Poloxamer 407 can be in a concentration between21 wt % and 40 wt % relative to the composition. In another embodiment,the Poloxamer 407 is in a concentration between 21 wt % and 30 wt %relative to the composition. In another embodiment, the Poloxamer 407 isin a concentration between 23 wt % and 29 wt % relative to thecomposition. In another embodiment, the Poloxamer 407 is in aconcentration between 23 wt % and 27 wt % relative to the composition.In another embodiment, the Poloxamer 407 is in a concentration of 25 wt% relative to the composition.

In some embodiments, the gelation temperature of the pharmaceuticalcomposition is between about 11° C. and about 32° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis between is between about 15° C. and about 30° C. In some embodiments,the gelation temperature of the pharmaceutical composition is betweenabout 20° C. and about 28° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is between about 24° C.and about 26° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 15° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 20° C.In some embodiments, the gelation temperature of the pharmaceuticalcomposition is about 24° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is about 26° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis about 28° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 30° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the amount of the Valproic acid inthe pharmaceutical composition is between least 2 wt % (weight Valproicacid/weight pharmaceutical composition) and 20 wt %. In someembodiments, the composition comprises at least 4 wt % Valproic acid. Inother embodiments, the composition comprises at least 8 wt % Valproicacid. In some embodiments, the composition comprises at least 12 wt %Valproic acid. In some embodiments, the composition comprises at least16 wt % Valproic acid. In still other embodiments, the compositioncomprises at least 20 wt % Valproic acid.

In other embodiments, the composition comprises about 4 wt % Valproicacid. In other embodiments, the composition comprises about 8 wt %Valproic acid. In other embodiments, the composition comprises about 12wt % Valproic acid. In further embodiments, the composition comprisesabout 16 wt % Valproic acid. In further embodiments, the compositioncomprises about 20 wt % Valproic acid.

A further aspect of the present disclosure provides a poloxamercontaining pharmaceutical composition having a gelation temperaturegreater than 10° C., the composition comprising

a) a compound of Formula III

or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 in weight percentage of more than 21% wt; and

c) Valproic acid;

wherein the pharmaceutical composition has a gelation temperature ofgreater than 10° C.

In some embodiments, the Poloxamer 407 can be in a concentration between21 wt % and 40 wt % relative to the composition. In another embodiment,the Poloxamer 407 is in a concentration between 21 wt % and 30 wt %relative to the composition. In another embodiment, the Poloxamer 407 isin a concentration between 23 wt % and 29 wt % relative to thecomposition. In another embodiment, the Poloxamer 407 is in aconcentration between 23 wt % and 27 wt % relative to the composition.In another embodiment, the Poloxamer 407 is in a concentration of 25 wt% relative to the composition.

In some embodiments, the gelation temperature of the pharmaceuticalcomposition is between about 11° C. and about 32° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis between is between about 15° C. and about 30° C. In some embodiments,the gelation temperature of the pharmaceutical composition is betweenabout 20° C. and about 28° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is between about 24° C.and about 26° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 15° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 20° C.In some embodiments, the gelation temperature of the pharmaceuticalcomposition is about 24° C. In some embodiments, the gelationtemperature of the pharmaceutical composition is about 26° C. In someembodiments, the gelation temperature of the pharmaceutical compositionis about 28° C. In some embodiments, the gelation temperature of thepharmaceutical composition is about 30° C. In some embodiments, thegelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the amount of the Valproic acid inthe pharmaceutical composition is between least 2 wt % (weight Valproicacid/weight pharmaceutical composition) and 20 wt %. In someembodiments, the composition comprises at least 4 wt % Valproic acid. Inother embodiments, the composition comprises at least 8 wt % Valproicacid. In some embodiments, the composition comprises at least 12 wt %Valproic acid. In some embodiments, the composition comprises at least16 wt % Valproic acid. In still other embodiments, the compositioncomprises at least 20 wt % Valproic acid.

In other embodiments, the composition comprises about 4 wt % Valproicacid. In other embodiments, the composition comprises about 8 wt %Valproic acid. In other embodiments, the composition comprises about 12wt % Valproic acid. In further embodiments, the composition comprisesabout 16 wt % Valproic acid. In further embodiments, the compositioncomprises about 20 wt % Valproic acid.

In any of the foregoing aspects and embodiments, the poloxamer can be ina concentration between 21 wt % and 40 wt % relative to the composition.In another embodiment, the poloxamer is in a concentration between 21 wt% and 30 wt % relative to the composition. In another embodiment, thepoloxamer is in a concentration between 23 wt % and 29 wt % relative tothe composition. In another embodiment, the poloxamer is in aconcentration between 23 wt % and 27 wt % relative to the composition.In another embodiment, the poloxamer is in a concentration of 25 wt %relative to the composition.

Poloxamers are commercially available (e.g., Pluronic® polyols areavailable from BASF Corporation). Alternatively, poloxamers can besynthesized by known techniques.

In one or more embodiments the viscosity of the composition at aboutbody temperature is substantially higher than the viscosity of thecomposition at 4° C. In some embodiments the viscosity of thecomposition at 4° C. is between 100 cp and 100,000 cp. In someembodiments the viscosity of the composition at 4° C. is between 100 cpand 10,000 cp. In some embodiments the viscosity of the composition at4° C. is between 100 cp and 1,000 cp. In some embodiments the viscosityof the composition at 37° C. is between 50,000 cp and 500,000 cp. Insome embodiments the viscosity of the composition at 37° C. is between100,000 cp and 300,000 cp. In some embodiments the viscosity of thecomposition at 37° C. is at least 10-fold greater than the viscosity ofthe composition at 4° C. In some embodiments the viscosity of thecomposition at 37° C. is at least 50-fold greater than the viscosity ofthe composition at 4° C.

In other embodiments, the carboxylic acid is in a concentration between20 and 160 mg/mL relative to the composition. In other embodiments, thecarboxylic acid is in a concentration of approximately 87.5 mg/mLrelative to the composition.

In some embodiments, it is desirable that the formulation besufficiently viscous that it remain in an area of administration for asuitable period of time, e.g., 1 min, 5 min, 10 min, 30 min., 1 hour, 5hours, 12, hours, 1 day, 2 days, 7 days. The desired area may beexternal to the eardrum, but in contact therewith, e.g., where it can bedesired that a layer of solution remain in contact with the eardrum(e.g., not flow out of ear) when the recipient subject is upright; evenif some of a viscous formulation exits the ear canal, a viscous film oflayer can, in some embodiments, be let in contact with the eardrum.

In some embodiments, the final formulation is a liquid at storagetemperature (4° C.), and formed a semisolid gel above its liquid-geltransition temperature (37° C.). In other embodiments, compositions havea viscosity of less than 100,000 centipoise (cps) at 25° C. Compositionsalso have a minimum yield stress that is sufficient for maintaining theformulation against the tympanic membrane. Yield stress is the amount offorce that causes a solid material to exhibit liquid-like behavior inthat it continues to deform with no further increase in stress. Minimumyield stress is dependent on the thickness of the applied gel, but isindependent of the geometry of the gel and the temperature of theenvironment. Minimum yield stress of a composition refers to an appliedgel having a thickness of 4 mm and a density of 1 g/L. Yield stress (Go)is represented as σ₀=ρgh, where ρ is density, g is the acceleration dueto gravity, and h is the layer thickness. Typically, minimum yieldstress is about 39 pascals (Pa).

Viscogenic agents are typically polymers or other chemical moieties thatincrease the viscosity of a fluid. Suitable viscogenic agents, whenincluded in a composition, allow the composition to transform from aliquid-like state (e.g., flowable) at 25° C. to a solid-like state(e.g., a gel) after contact with the tympanic membrane, and can benon-biodegradable, (e.g., not broken down by chemicals or enzymesnaturally present in a mammal, or biodegradable). Compositions includean amount of viscogenic agent effective to yield a viscosity of thecomposition of less than 100,000 cps at 25° C. (e.g., less than 90,000,60,000, 30,000, 20,000, or 10,000 cps) and, generally, a minimum yieldstress of 39 Pa after application to the tympanic membrane. Typically, acomposition includes 0.05 to 50% of a viscogenic agent (e.g., 0.15 to25, 5 to 45, 10 to 40, 12 to 37, 15 to 35, 17 to 33, or 20 to 30% of aviscogenic agent).

Exemplary viscogenic agents include gellan (GELRITE or KELCOGEL),CARBOPOL 940 with hydroxypropylmethylcellulose (HPMC), N-isopropylacrylamide (NiPAAm) with sodium acrylate and n-N-alkylacrylamide,polyacrylic acid with polyethylene glycol (PEG) or polymethacryhc acidwith PEG, cellulose acetate hydrogen phthalate latex (CAP), sodiumalginate, and nonionic surfactants such as poloxamers (PLURIONIC) andpolyoxamine (TETRONIC) reversible temperature-dependent gelling systems.

Among other things, the compositions provided herein are useful for theprophylaxis and/or treatment of acute and chronic ear disease andhearing loss, dizziness and balance problems especially of suddenhearing loss, acoustic trauma, hearing loss due to chronic noiseexposure, presbycusis, trauma during implantation of the inner earprosthesis (insertion trauma), dizziness due to diseases of the innerear area, dizziness related and/or as a symptom of Meniere's disease,vertigo related and/or as a symptom of Meniere's disease, tinnitus, andhearing loss due to antibiotics and cytostatics and other drugs.

Stem cells exhibit an extraordinary ability to generate multiple celltypes in the body. Besides embryonic stem cells, tissue specific stemcells serve a critical role during development as well as in homeostasisand injury repair in the adult. Stem cells renew themselves throughproliferation as well as generate tissue specific cell types throughdifferentiation. The characteristics of different stem cells vary fromtissue to tissue, and are determined by their intrinsic genetic andepigenetic status. However, the balance between self-renewal anddifferentiation of different stem cells are all stringently controlled.Uncontrolled self-renewal may lead to overgrowth of stem cells andpossibly tumor formation, while uncontrolled differentiation may exhaustthe stem cell pool, leading to an impaired ability to sustain tissuehomeostasis. Thus, stem cells continuously sense their environment andappropriately respond with proliferation, differentiation or apoptosis.It would be desirable to drive regeneration by controlling the timingand extent of stem cell proliferation and differentiation. Controllingthe proliferation with small molecules that are cleared over time wouldallow for control of the timing and extent of stem cell proliferationand differentiation. Remarkably, tissue stem cells from differenttissues share a limited number of signaling pathways for the regulationof their self-renewal and differentiation, albeit in a very contextdependent manner. Some of these pathways are the Wnt and GSK3-betapathways.

Lgr5 is expressed across a diverse range of tissues and has beenidentified as a biomarker of adult stem cells in a variety of tissuessuch as the gut epithelia (Barker et al. 2007), kidney, hair follicle,and stomach (Barker et al, 2010; Haegebarth & Clevers, 2009). Forexample, it was first published in 2011, that mammalian inner ear haircells are derived from LGR5⁺ cells (Chai et al, 2011, Shi et al. 2012).Lgr5 is a known component of the Wnt/beta-catenin pathway, which hasbeen shown to play major roles in differentiation, proliferation, andinducing stem cell characteristics (Barker et al. 2007).

Permanent damage to the hair cells of the inner ear results insensorineural hearing loss, leading to communication difficulties in alarge percentage of the population. Hair cells are the receptor cellsthat transduce the acoustic stimulus. Regeneration of damaged hair cellswould provide an avenue for the treatment of a condition that currentlyhas no therapies other than prosthetic devices. Although hair cells donot regenerate in the mammalian cochlea, new hair cells in lowervertebrates are generated from epithelial cells, called supportingcells, that surround hair cells.

Prior work has focused on transdifferentiation of supporting cells intohair cells through activation or forced expression of genes that lead tohair cell formation, with a particular focus on mechanisms to enhanceexpression of Atoh1 (Bermingham et al., 1999; Zheng and Gao, 2000;Izumikawa et al., 2005; Mizutari et al., 2013). Interestingly, cellstransduced with Atoh1 vectors have been shown to acquire vestibularphenotypes (Kawamoto et al., 2003; Huang et al., 2009; Yang et al.,2012, 2013), and lack complete development. As mentioned, upregulatingAtoh1 via gene insertion has been shown to create non-cochlear celltypes that behave in a manner that is not found within the nativecochlea. In addition, these methods increase hair cell numbers butdecrease supporting cell numbers. Since supporting cells are known tohave specialized roles (Ramirez-Camancho 2006, Dale and Jagger 2010),loss of these cells could create problems in proper cochlear function.

In one aspect the present disclosure provides compositions useful forcontrolled proliferation of stem cells comprising administering to acell population an effective amount of a composition herein described.

Among the various aspects of the present disclosure, therefore, may benoted a method for activating the Wnt pathway in a cell population toincrease the capacity of the population for self-renewal, i.e., thecapacity for repeated generation of daughter cells with equivalentproliferation and ‘cell fate specification’ potential, anddifferentiation, i.e., the capacity for generation of daughter cellsspecified for differentiation. In one embodiment, the cell population isa cochlear supporting cell population. The Wnt pathway may also beactivated upstream of the c-myc gene in members of the population andwithout any genetic modification of the population. Further, the Wntpathway can also be activated by small molecules that transiently inducesuch activity. Additionally, the supporting cell population includessupporting cells that are LGR5⁺ and endogenous to the Organ of Corti.

In certain embodiments a composition includes a Notch agonist or an HDACinhibitor selected such as Valproic acid or a prodrug, ester, salt form,or amide thereof. Optionally, the composition also includes a StemnessDriver. Optionally, the Stemness Driver is a Wnt agonist. In someembodiments, the Wnt agonist is a GSK3 inhibitor. In some embodimentsthe Wnt agonist is CHIR99021, LY2090314, AZD 1080, or GSK3 InhibitorXXII.

In certain embodiments, therefore, the present disclosure providescompositions that have the capacity to induce self-renewal of apopulation of supporting cells. These compositions have the capacity toactivate pathways and mechanisms that are known to be involved ininducing stem cell properties, such as those used to create “inducedpluripotent stem cells”. These pathways are activated with smallmolecules. For example, a composition when applied in vitro to asupporting cell population may induce the population to proliferate to ahigh degree and in high purity in a Stem Cell Proliferation Assay, andalso allow the population to differentiate into a high purity populationof hair cells in a Stem Cell Differentiation Assay. In one suchembodiment, the composition induces and maintains stem cell propertiesby proliferating to produce stem cell that can divide for manygenerations and maintain the ability to have a high proportion of theresulting cells differentiate into hair cells. Further, theproliferating stem cells express stem cell markers which may include oneor more of Lgr5, Sox2, Opem1, Phex, lin28, Lgr6, cyclin D1, Msx1, Myb,Kit, Gdnf3, Zic3, Dppa3, Dppa4, Dppa5, Nanog, Esrrb, Rex1, Dnmt3a,Dnmt3b, Dnmt31, Utf1, Tcl1, Oct4, Klf4, Pax6, Six2, Zic1, Zic2, Otx2,Bmi1, CDX2, STAT3, Smad1, Smad2, smad2/3, smad4, smad5, and smad7.

In certain embodiments, the disclosure provides a method of andcompositions for generating hair cells, the method comprising:administering or causing to be administered to a stem cell population(e.g., of an in vitro, ex vivo, or in vivo sample/subject) a compositioncomprising a GSK3-beta inhibitor (or a derivative or pharmaceuticallyacceptable salt thereof) and/or Wnt agonist (or a derivative orpharmaceutically acceptable salt thereof), thereby proliferating stemcells in the stem cell population and resulting in an expandedpopulation of stem cells; and exposing the expanded population of stemcells to a GSK3-beta inhibitor (or a derivative or pharmaceuticallyacceptable salt thereof) and/or a Wnt agonist (or a derivative orpharmaceutically acceptable salt thereof), thereby facilitatinggeneration of inner ear hair cells from the expanded population of stemcells.

In certain embodiments, the disclosure provides methods for preventingand treating auditory dysfunction. For example, in certain embodiments,the disclosure provides methods for preventing or treating auditoryimpairments or restoring and improving hearing in a subject comprisingadministering to said subject an effective amount of a compositionherein describe.

The methods and compositions of the present disclosure allow greater andthus more effective dosing with these ototoxicity-inducingpharmaceutical drugs, while concomitantly preventing or reducingototoxic effects caused by these drugs. The methods of the presentdisclosure provide a safe, effective, and prolonged means forprophylactic or curative treatment of hearing impairments related toinner ear tissue damage, loss, or degeneration, particularly sound oraging-induced, and ototoxin-induced, and particularly involving innerear hair cells. In certain embodiments, the present disclosure providescompositions and methods that address one or more of these or othergoals.

This disclosure relates to compositions useful in methods for inducing,promoting, or enhancing the growth, proliferation, or regeneration ofinner ear tissue, for example, inner ear supporting cells and/or innerear hair cells.

The present disclosure relates to methods and compositions to activatethe Wnt pathway or to inhibit GSK3-beta activity.

In another aspect the present disclosure provides to compositions andmethods for controlled proliferation of stem cells comprisingadministering to a cell population an effective amount of a compositionprovided herein.

In another aspect the present disclosure relates to compositions andmethods to prevent, reduce or treat the incidence and/or severity ofdisorders or diseases associated with absence or lack of certain tissuecells. In one aspect the present disclosure relates to compositions andmethods to prevent, reduce or treat the incidence and/or severity ofinner ear disorders and hearing impairments involving inner ear tissue,particularly inner ear hair cells, their progenitors, and optionally,the stria vascularis, and associated auditory nerves. Of particularinterest are those conditions that lead to permanent hearing loss wherereduced number of hair cells may be responsible and/or decreased haircell function. Also of interest are those arising as an unwantedside-effect of ototoxic therapeutic drugs including cisplatin and itsanalogs, aminoglycoside antibiotics, salicylate and its analogs, or loopdiuretics. In certain embodiments, the present disclosure relates toinducing, promoting, or enhancing the growth, proliferation orregeneration of inner ear tissue, particularly inner ear supportingcells and hair cells.

When cochlea supporting cell populations are treated with thecompositions described herein, whether the population is in vivo or invitro, the treated supporting cells exhibit stem-like behavior in thatthe treated supporting cells have the capacity to proliferate anddifferentiate and, more specifically, differentiate into cochlear haircells. Alternatively, the composition induces and maintains thesupporting cells to produce daughter stem cells that can divide for manygenerations and maintain the ability to have a high proportion of theresulting cells differentiate into hair cells. In certain embodiments,the proliferating stem cells express stem cell markers which may includeLgr5, Sox2, Opem1, Phex, lin28, Lgr6, cyclin D1, Msx1, Myb, Kit, Gdnf3,Zic3, Dppa3, Dppa4, Dppa5, Nanog, Esrrb, Rex1, Dnmt3a, Dnmt3b, Dnmt31,Utf1, Tcl1, Oct4, Klf4, Pax6, Six2, Zic1, Zic2, Otx2, Bmi1, CDX2, STAT3,Smad1, Smad2, smad2/3, smad4, smad5, and/or smad7.

In some embodiments, the method of the present disclosure may be used tomaintain, or even transiently increase stemness (i.e., self-renewal) ofa pre-existing supporting cell population prior to significant hair cellformation. In some embodiments, the pre-existing supporting cellpopulation comprises inner pillar cells, outer pillar cells, innerphalangeal cells, Deiter cells, Hensen cells, Boettcher cells, and/orClaudius cells. Morphological analyses with immunostaining (includingcell counts) and lineage tracing across a Representative MicroscopySamples may be used to confirm expansion of one or more of thesecell-types. In some embodiments, the pre-existing supporting cellscomprise Lgr5⁺ cells. Morphological analyses with immunostaining(including cell counts) and qPCR and RNA hybridization may be used toconfirm Lgr5 upregulation amongst the cell population.

Advantageously, the methods of the present disclosure achieve thesegoals without the use of genetic manipulation. Germ-line manipulationused in many academic studies is not a therapeutically desirableapproach to treating hearing loss. In general, the therapy involves theadministration of a small molecule, peptide, antibody, or othernon-nucleic acid molecule or nucleic acid delivery vector unaccompaniedby gene therapy. In certain embodiments, the therapy involves theadministration of a small organic molecule. Alternatively, hearingprotection or restoration is achieved through the use of a (non-genetic)therapeutic that is injected in the middle ear and diffuses into thecochlea.

The cochlea relies heavily on all present cell types, and theorganization of these cells is important to their function. Assupporting cells play an important role in neurotransmitter cycling andcochlear mechanics. Thus, maintaining a rosette patterning within theorgan of Corti may be important for function. Cochlear mechanics of thebasilar membrane activate hair cell transduction. Due to the highsensitivity of cochlear mechanics, it is also desirable to avoid massesof cells. In all, maintaining proper distribution and relation of haircells and supporting cells along the basilar membrane, even afterproliferation, is likely a desired feature for hearing as supportingcell function and proper mechanics is necessary for normal hearing.

In one embodiment of the present disclosure, the cell density of haircells in a cochlear cell population is expanded in a manner thatmaintains, or even establishes, the rosette pattern characteristic ofcochlear epithelia.

In accordance with one aspect of the present disclosure, the celldensity of hair cells may be increased in a population of cochlear cellscomprising both hair cells and supporting cells. The cochlear cellpopulation may be an in vivo population (i.e., comprised by the cochlearepithelium of a subject) or the cochlear cell population may be an invitro (ex vivo) population. If the population is an in vitro population,the increase in cell density may be determined by reference to aRepresentative Microscopy Sample of the population taken prior andsubsequent to any treatment. If the population is an in vivo population,the increase in cell density may be determined indirectly by determiningan effect upon the hearing of the subject with an increase in hair celldensity correlating to an improvement in hearing.

In one embodiment, supporting cells placed in a Stem Cell ProliferationAssay in the absence of neuronal cells form ribbon synapses.

In a native cochlea, patterning of hair cells and supporting cellsoccurs in a manner parallel to the basilar membrane. In one embodimentof the present disclosure, the proliferation of supporting cells in acochlear cell population is expanded in a manner that the basilarmembrane characteristic of cochlear epithelia.

In one embodiment, the number of supporting cells in an initial cochlearcell population is selectively expanded by treating the initial cochlearcell population with a composition comprising the composition comprisingthe composition comprising the salt of Formula I to form an intermediatecochlear cell population and wherein the ratio of supporting cells tohair cells in the intermediate cochlear cell population exceeds theratio of supporting cells to hair cells in the initial cochlear cellpopulation. The expanded cochlear cell population may be, for example,an in vivo population, an in vitro population or even an in vitroexplant. In one such embodiment, the ratio of supporting cells to haircells in the intermediate cochlear cell population exceeds the ratio ofsupporting cells to hair cells in the initial cochlear cell population.For example, in one such embodiment the ratio of supporting cells tohair cells in the intermediate cochlear cell population exceeds theratio of supporting cells to hair cells in the initial cochlear cellpopulation by a factor of 1.1. By way of further example, in one suchembodiment the ratio of supporting cells to hair cells in theintermediate cochlear cell population exceeds the ratio of supportingcells to hair cells in the initial cochlear cell population by a factorof 1.5. By way of further example, in one such embodiment the ratio ofsupporting cells to hair cells in the intermediate cochlear cellpopulation exceeds the ratio of supporting cells to hair cells in theinitial cochlear cell population by a factor of 2. By way of furtherexample, in one such embodiment the ratio of supporting cells to haircells in the intermediate cochlear cell population exceeds the ratio ofsupporting cells to hair cells in the initial cochlear cell populationby a factor of 3. In each of the foregoing embodiments, the capacity ofa composition of the present disclosure to expand a cochlear cellpopulation as described in this paragraph may be determined by means ofa Stem Cell Proliferation Assay.

In one embodiment, the number of stem cells in a cochlear cellpopulation is expanded to form an intermediate cochlear cell populationby treating a cochlear cell population with a composition comprising thecomposition comprising the composition comprising the salt of Formula Iwherein the cell density of stem cells in the intermediate cochlear cellpopulation exceeds the cell density of stem cells in the initialcochlear cell population. The treated cochlear cell population may be,for example, an in vivo population, an in vitro population or even an invitro explant. In one such embodiment, the cell density of stem cells inthe treated cochlear cell population exceeds the cell density of stemcells in the initial cochlear cell population by a factor of at least1.1. For example, in one such embodiment the cell density of stem cellsin the treated cochlear cell population exceeds the cell density of stemcells in the initial cochlear cell population by a factor of at least1.25. For example, in one such embodiment the cell density of stem cellsin the treated cochlear cell population exceeds the cell density of stemcells in the initial cochlear cell population by a factor of at least1.5. By way of further example, in one such embodiment the cell densityof stem cells in the treated cochlear cell population exceeds the celldensity of stem cells in the initial cochlear cell population by afactor of at least 2. By way of further example, in one such embodimentthe cell density of stem cells in the treated cochlear cell populationexceeds the cell density of stem cells in the initial cochlear cellpopulation by a factor of at least 3. In vitro cochlear cell populationsmay expand significantly more than in vivo populations; for example, incertain embodiments the cell density of stem cells in an expanded invitro population of stem cells may be at least 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 2,000 or even 3,000 times greater than the cell density ofthe stem cells in the initial cochlear cell population. In each of theforegoing embodiments, the capacity of a composition of the presentdisclosure to expand a cochlear cell population as described in thisparagraph may be determined by means of a Stem Cell Proliferation Assay.

In accordance with one aspect of the present disclosure, a cochleasupporting cell population is treated with a composition comprising thecomposition comprising the salt of Formula I to increase the Lgr5activity of the population. For example, in one embodiment thecomposition comprising the salt of Formula I has the capacity toincrease and maintain the Lgr5 activity of an in vitro population ofcochlea supporting cells by factor of at least 1.2. By way of furtherexample, in one such embodiment the composition has the capacity toincrease the Lgr5 activity of an in vitro population of cochleasupporting cells by factor of 1.5. By way of further example, in onesuch embodiment the composition has the capacity to increase the Lgr5activity of an in vitro population of cochlea supporting cells by factorof 2, 3, 5 10, 100, 500, 1,000, 2,000 or even 3,000. Increases in Lgr5activity may also be observed for in vivo populations but the observedincrease may be somewhat more modest. For example, in one embodiment thecomposition has the capacity to increase the Lgr5 activity of an in vivopopulation of cochlea supporting cells by at least 5%. By way of furtherexample, in one such embodiment the composition has the capacity toincrease the Lgr5 activity of an in vivo population of cochleasupporting cells by at least 10%. By way of further example, in one suchembodiment the composition has the capacity to increase the Lgr5activity of an in vivo population of cochlea supporting cells by atleast 20%. By way of further example, in one such embodiment thecomposition has the capacity to increase the Lgr5 activity of an in vivopopulation of cochlea supporting cells by at least 30%. In each of theforegoing embodiments, the capacity of the composition for such anincrease in Lgr5 activity may be demonstrated, for example, in an Invitro Lgr5⁺ Activity Assay and in an in vivo population may bedemonstrated, for example, in an In vivo Lgr5⁺ Activity Assay, asmeasured by isolating the organ and performing morphological analysesusing immunostaining, endogenous fluorescent protein expression of Lgr5(e.g., Lgr5, Sox2), and qPCR for Lgr5.

In addition to increasing the Lgr5 activity of the population, thenumber of Lgr5⁺ supporting cells in a cochlea cell population may beincreased by treating a cochlea cell population containing Lgr5+supporting cells (whether in vivo or in vitro) with a compositioncomprising the composition comprising the salt of Formula I. In general,the cell density of the stem/progenitor supporting cells may expandrelative to the initial cell population via one or more of severalmechanisms. For example, in one such embodiment, newly generated Lgr5⁺supporting cells may be generated that have increased stem cellpropensity (i.e., greater capacity to differentiate into hair cell). Byway of further example, in one such embodiment no daughter Lgr5⁺ cellsare generated by cell division, but pre-existing Lgr5⁺ supporting cellsare induced to differentiate into hair cells. By way of further example,in one such embodiment no daughter cells are generated by cell division,but Lgr5⁻ supporting cells are activated to a greater level of Lgr5activity and the activated supporting cells are then able todifferentiate into hair cells. Regardless of the mechanism, in oneembodiment the composition of the present disclosure has the capacity toincrease the cell density of Lgr5⁺ supporting cells in an in vitroisolated cell population of cochlea supporting cells by factor of atleast 5. By way of further example, in one such embodiment thecomposition has the capacity to increase the cell density of Lgr5⁺supporting cells in an in vitro population of cochlea supporting cellsby factor of at least 10. By way of further example, in one suchembodiment the composition has the capacity to increase the cell densityof Lgr5⁺ supporting cells in an in vitro population of cochleasupporting cells by factor of at least 100, at least 500, at least 1,000or even at least 2,000. Increases in the cell density of Lgr5⁺supporting cells may also be observed for in vivo populations but theobserved increase may be somewhat more modest. For example, in oneembodiment the composition has the capacity to increase the cell densityof Lgr5+ supporting cells in an in vivo population of cochlea supportingcells by at least 5%. By way of further example, in one such embodimentthe composition has the capacity to increase the cell density of Lgr5⁺supporting cells in an in vivo population of cochlea supporting cells byat least 10%. By way of further example, in one such embodiment thecomposition has the capacity to increase the cell density of Lgr5⁺supporting cells in an in vivo population of cochlea supporting cells byat least 20%. By way of further example, in one such embodiment thecomposition has the capacity to increase the cell density of Lgr5⁺supporting cells in an in vivo population of cochlea supporting cells byat least 30%. The capacity of the composition for such an increase inLgr5+ supporting cells in an in vitro population may be demonstrated,for example, in a Stem Cell Proliferation Assay or in an appropriate invivo assay. In one embodiment, a composition of the present disclosurehas the capacity to increase the number of Lgr5⁺ cells in the cochlea byinducing expression of Lgr5 in cells with absent or low detection levelsof the protein, while maintaining Native Morphology. In one embodiment,a composition of the present disclosure has the capacity to increase thenumber of Lgr5⁺ cells in the cochlea by inducing expression of Lgr5 incells with absent or low detection levels of the protein, whilemaintaining Native Morphology and without producing Cell Aggregates.

In addition to increasing the cell density of Lgr5⁺ supporting cells, inone embodiment the method of the present disclosure has the capacity toincrease the ratio of Lgr5⁺ cells to hair cells in a cochlear cellpopulation. In one embodiment, the number of Lgr5⁺ supporting cells inan initial cochlear cell population is selectively expanded by treatingthe initial cochlear cell population with a composition of the presentdisclosure to form an expanded cell population and wherein the number ofLgr5⁺ supporting cells in the expanded cochlear cell population at leastequals the number of hair cells. The expanded cochlear cell populationmay be, for example, an in vivo population, an in vitro population oreven an in vitro explant. In one such embodiment, the ratio of Lgr5⁺supporting cells to hair cells in the expanded cochlear cell populationis at least 1:1. For example, in one such embodiment the ratio of Lgr5⁺supporting cells to hair cells in the expanded cochlear cell populationis at least 1.5:1. By way of further example, in one such embodiment theratio of Lgr5⁺ supporting cells to hair cells in the expanded cochlearcell population is at least 2:1. By way of further example, in one suchembodiment the ratio of Lgr5⁺ supporting cells to hair cells in theexpanded cochlear cell population is at least 3:1. By way of furtherexample, in one such embodiment the ratio of Lgr5⁺ supporting cells tohair cells in the expanded cochlear cell population is at least 4:1. Byway of further example, in one such embodiment the ratio of Lgr5⁺supporting cells to hair cells in the expanded cochlear cell populationis at least 5:1. In each of the foregoing embodiments, the capacity ofthe composition of the present disclosure to expand a cochlear cellpopulation as described in this paragraph may be determined by means ofa Stem Cell Proliferation Assay.

In certain embodiments, the method increases the fraction of the Lgr5⁺cells to total cells on the sensory epithelium by at least 10%, 20%,50%, 100%, 250% 500%, 1,000% or 5000%.

In certain embodiments, the method increases the Lgr5⁺ cells until theybecome at least 10, 20, 30, 50, 70, or 85% of the cells on the sensoryepithelium, e.g., the Organ of Corti.

In general, excessive proliferation of supporting cells in the cochleais avoided. In one embodiment, the method of the present disclosure hasthe capacity to expand a cochlear cell population without creating aprotrusion of new cells beyond the native surface of the cochlea, e.g.,a Cell Aggregate. In some embodiments, 30 days after placing acomposition comprising the composition comprising the salt of Formula Ion the round or oval window membrane, the cochlear tissue has NativeMorphology. In some embodiments, 30 days after placing the compositionon the round or oval window membrane, the cochlear tissue has NativeMorphology and lacks Cell Aggregates. In some embodiments, 30 days afterplacing the composition on the round or oval window membrane, thecochlear tissue has Native Morphology and at least 10, 20, 30, 50, 75,90, 95, 98, or even at least 99% of the Lgr5⁺ cells in the Organ ofCorti are not part of Cell Aggregates.

In addition to expanding supporting cell populations, generally, andLgr5⁺ supporting cells, specifically, as described above, the method ofthe present disclosure has the capacity to maintain, in the daughtercells, the capacity to differentiate into hair cells. In in vivopopulations, the maintenance of this capacity may be indirectly observedby an improvement in a subject's hearing. In in vitro populations, themaintenance of this capacity may be directly observed by an increase inthe number of hair cells relative to a starting population or indirectlyby measuring LGR5 activity, SOX2 activity or one or more of the otherstem cell markers identified elsewhere herein.

In one embodiment, the capacity of the method to increase the stemnessof a population of cochlear supporting cells, in general, or apopulation of Lgr5+ supporting cells, in particular, may be correlatedwith an increase of Lgr5 activity of an in vitro population of isolatedLgr5⁺ cells as determined by an Lgr5 Activity Assay. As previouslynoted, in one such embodiment, the composition has the capacity toincrease the Lgr5 activity of stem cells in the intermediate cellpopulation by a factor of 5 on average relative to the Lgr5 activity ofthe cells in the initial cell population. By way of further example, inone such embodiment the method has the capacity to increase the Lgr5activity of the stem cells genes in the intermediate cell population bya factor of 10 relative to the Lgr5 activity of the cells in the initialcell population. By way of further example, in one such embodiment themethod has the capacity to increase the Lgr5 activity of the stem cellsin the intermediate cell population by a factor of 100 relative to theLgr5 activity of the cells in the initial cell population. By way offurther example, in one such embodiment the method has the capacity toincrease the Lgr5 activity of the stem cells in the intermediate cellpopulation by a factor of 1000 relative to the Lgr5 activity of thecells in the initial cell population. In each of the foregoingembodiments, the increase in the activity of stem cells in the cellpopulation may be determined in vitro by immunostaining or endogenousfluorescent protein expression for target genes and analysis of theirrelative intensities via imaging analysis or flowcytometry, or usingqPCR for target stem cell genes. The identity of the resulting stem cellpopulation may optionally be further determined by stem cell assaysincluding stem cell marker expression assay, colony forming assay,self-renewal assay and differentiation assay as defined in Stem cellassay.

In some embodiments, the method applied to an adult mammal produces apopulation of adult mammalian Lgr5⁺ cells that are in S-phase.

In one embodiment, after applying the composition comprising thecomposition comprising the salt of Formula I to the round or oval windowof a mouse, the in vivo Lgr5⁺ Activity of a cell population in the Organof Corti increases 1.3×, 1.5×, up to 20× over baseline for a populationthat has not been exposed to the composition. In some embodiments,applying the composition to the round or oval window of a mouseincreases the average In vivo Lgr5⁺ Activity for cells in the Organ ofCorti is increased 1.3×, 1.5×, up to 20× over baseline for a populationthat has not been exposed to the composition.

In certain embodiments, the method increases the Lgr5⁺ cells until theybecome at least 10%, 7.5%, 10%, up to 100% of the supporting cellpopulation by number.

In certain embodiments, the composition has the capacity to increase thepercentage of Lgr5⁺ cell in a cochlea by 5%, 10%, 25%, 50%, or 80%.

In certain embodiments, the stem cell population is of an in vivosubject, and the method is a treatment for hearing loss and/orvestibular dysfunction (e.g., wherein the generation of inner ear haircells from the expanded population of stem cells results in partial orfull recovery of hearing loss and/or improved vestibular function). Incertain embodiments, the stem cell population is of an in vivo subject,and the method further comprises delivering a drug to the subject (e.g.,for treatment of a disease and/or disorder unrelated to hearing lossand/or vestibular dysfunction) at a higher concentration than a knownsafe maximum dosage of the drug for the subject (e.g., the known safemaximum dosage if delivered in the absence of the generation of innerear hair cells resulting from the method) (e.g., due to a reduction orelimination of a dose-limiting ototoxicity of the drug).

In certain embodiments, the method further comprises performing highthroughput screening using the generated inner ear hair cells. Incertain embodiments, the method comprises using the generated inner earhair cells to screen molecules for toxicity against inner ear haircells. In certain embodiments, the method comprises using the generatedinner ear hair cells to screen molecules for ability to improve survivalof inner ear hair cells (e.g., inner ear hair cells exposed to saidmolecules).

In another aspect, the disclosure is directed to a method of producingan expanded population of stem cells, the method comprising:administering or causing to be administered to a stem cell population(e.g., of an in vitro, ex vivo, or in vivo sample/subject) a compositioncomprising the composition comprising the salt of Formula I.

In certain embodiments, the administering step is carried out byperforming one or more injections into the ear (e.g., transtympanicallyinto the middle ear and/or inner ear).

In certain embodiments, the administering step comprises administeringthe GSK3-beta inhibitor and/or Wnt agonist in a sustained manner.

In certain embodiments, the stem cells are inner ear stem cells and/orsupporting cells.

In certain embodiments, the method further comprises performing highthroughput screening using the generated expanded population of stemcells. In certain embodiments, the method further comprises using thegenerated stem cells to screen molecules for toxicity against stem cellsand/or their progeny. In certain embodiments, the method comprises usingthe generated stem cells to screen molecules for ability to improvesurvival of stem cells and/or their progeny.

In another aspect, the disclosure is directed to a method of treating asubject who has, or is at risk of developing, hearing loss and/orvestibular dysfunction, the method comprising: identifying a subject whohas experienced, or is at risk for developing, hearing loss and/orvestibular dysfunction, administering or causing to be administered acomposition comprising any of the compositions or compounds providedherein.

In certain embodiments, the stem cell population comprises Lgr5⁺ cells.In certain embodiments, the stem cell population comprises post-natalcells. In certain embodiments, the stem cell population comprisesepithelial stem cells. In certain embodiments, stem cells includeprogenitor cells.

In certain embodiments, the step of administering is carried out byperforming one or more injections into the ear (e.g., transtympanicallyinto the middle ear and/or inner ear).

In another aspect, the disclosure is directed to a method of generatinginner ear hair cells, the method comprising: proliferating stem cells inan initial stem cell population (e.g., of an in vitro, ex vivo, or invivo sample/subject), resulting in an expanded population of stem cells(e.g., such that the expanded population is a factor of at least 1.25,1.5, 1.75, 2, 3, 5, 10, or 20 greater than the initial stem cellpopulation); and facilitating generation of inner ear hair cells fromthe expanded population of stem cells.

In another aspect, the disclosure is directed to a method of generatinginner ear hair cells, the method comprising administering a compositioncomprising the composition comprising the salt of Formula I (e.g., in apharmaceutically acceptable form (e.g., salt)) to a cell population inan inner ear of a subject, thereby facilitating generation of inner earhair cells.

In another aspect, the disclosure is directed to a method of generatinginner ear hair cells, the method comprising: proliferating post-natalLGR5+ cells in an initial population (e.g., of an in vitro, ex vivo, orin vivo sample/subject), resulting in an expanded population of LGR5+cells (e.g., such that the expanded population is a factor of at least1.25, 1.5, 1.75, 2, 3, 5, 10, or 20 greater than the initial stem cellpopulation), said expanded population of LGR5+ cells resulting ingeneration of inner ear hair cells. In certain embodiments, stem cellsinclude progenitor cells.

In another aspect, the disclosure is directed to a method of treating adisease or disorder, the method comprising: proliferating post-natalLgr5⁺ epithelial cells in an initial population of a subject (in vivo),resulting in an expanded population of Lgr5+ epithelial cells (e.g.,such that the expanded population is a factor of at least 1.25, 1.5,1.75, 2, 3, 5, 10, or 20 greater than the initial post-natal Lgr5⁺epithelial cell population).

In some embodiments, Lgr5⁺ cells are differentiated into hair cells.

In some embodiments of the compositions described herein, the Wntagonist, GSK-3 alpha inhibitor or GSK-3 beta inhibitor is used at aconcentration of about 0.01 uM to about 1,000 mM and optionally incombination with other agents. In other embodiments, the Wnt agonist,GSK-3 alpha inhibitor or GSK-3 beta inhibitor is used at a concentrationof about 0.1 uM to about 1,000 mM and optionally in combination withother agents. In other embodiments, the Wnt agonist, GSK-3 alphainhibitor or GSK-3 beta inhibitor is used at a concentration of about 1uM to about 100 mM and optionally in combination with other agents. Inother embodiments, the Wnt agonist, GSK-3 alpha inhibitor or GSK-3 betainhibitor is used at a concentration of about 10 uM to about 10 mM andoptionally in combination with other agents. In other embodiments, theWnt agonist, GSK-3 alpha inhibitor or GSK-3 beta inhibitor is used at aconcentration of about 1 uM to about 10 uM and optionally in combinationwith other agents. In other embodiments, the Wnt agonist, GSK-3 alphainhibitor or GSK-3 beta inhibitor is used at a concentration of about 10uM to about 100 uM and optionally in combination with other agents. Inother embodiments, the Wnt agonist, GSK-3 alpha inhibitor or GSK-3 betainhibitor is used at a concentration of about 100 uM to about 1000 uMand optionally in combination with other agents. In other embodiments,the Wnt agonist, GSK-3 alpha inhibitor or GSK-3 beta inhibitor is usedat a concentration of about 1 mM to about 10 mM and optionally incombination with other agents. In other embodiments, the Wnt agonist,GSK-3 alpha inhibitor or GSK-3 beta inhibitor is used at a concentrationof about 10 mM to about 100 mM and optionally in combination with otheragents.

In some embodiments of the compositions described herein, the GSK-3inhibitor is CHIR99021 used at a concentration of about 1 uM to about1,000 mM and optionally in combination with other agents. In otherembodiments, the GSK-3 inhibitor is CHIR99021 used at a concentration ofabout 10 uM to about 100 mM and optionally in combination with otheragents. In other embodiments, the GSK-3 inhibitor is CHIR99021 used at aconcentration of about 100 uM to about 100 mM and optionally incombination with other agents. In other embodiments, the GSK-3 inhibitoris CHIR99021 used at a concentration of about 1 mM to about 10 mM andoptionally in combination with other agents.

In some embodiments of the compositions described herein, the GSK-3inhibitor is LY2090314 used at a concentration of about 0.01 uM to about1,000 mM and optionally in combination with other agents. In otherembodiments, the GSK-3 inhibitor is LY2090314 used at a concentration ofabout 0.1 uM to about 10 mM and optionally in combination with otheragents. In other embodiments, the GSK-3 inhibitor is LY2090314 used at aconcentration of about 1 uM to about 1 mM and optionally in combinationwith other agents. In other embodiments, the GSK-3 inhibitor isLY2090314 used at a concentration of about 1 uM to about 100 uM andoptionally in combination with other agents. In other embodiments, theGSK-3 inhibitor is LY2090314 used at a concentration of about 10 uM andoptionally in combination with other agents.

In some embodiments of the compositions described herein, the GSK-3inhibitor is GSK inhibitor XXII used at a concentration of about 0.1 uMto about 1,000 mM and optionally in combination with other agents. Inother embodiments, the GSK-3 inhibitor is GSK inhibitor XXII used at aconcentration of about 1 uM to about 100 mM and optionally incombination with other agents. In other embodiments, the GSK-3 inhibitoris GSK inhibitor XXII used at a concentration of about 10 uM to about 10mM and optionally in combination with other agents. In otherembodiments, the GSK-3 inhibitor is GSK inhibitor XXII used at aconcentration of about 100 uM to about 10 mM and optionally incombination with other agents. In other embodiments, the GSK-3 inhibitoris GSK inhibitor XXII used at a concentration of about 100 uM to about 1mM and optionally in combination with other agents.

In some embodiments of the compositions described herein, the GSK-3inhibitor is AZD 1080 used at a concentration of about 0.1 uM to about1,000 mM and optionally in combination with other agents. In otherembodiments, the GSK-3 inhibitor is AZD 1080 used at a concentration ofabout 1 uM to about 1,000 mM and optionally in combination with otheragents. In other embodiments, the GSK-3 inhibitor is AZD 1080 used at aconcentration of about 10 uM to about 100 mM and optionally incombination with other agents. In other embodiments, the GSK-3 inhibitoris AZD 1080 used at a concentration of about 100 uM to about 10 mM andoptionally in combination with other agents. In other embodiments, theGSK-3 inhibitor is AZD 1080 used at a concentration of about 1 mM toabout 10 mM and optionally in combination with other agents.

In some embodiments of the compositions described herein, the HDACinhibitor is used at a concentration of about 0.01 uM to about 100,000mM and optionally in combination with other agents. In otherembodiments, the HDAC inhibitor is used at a concentration of about 1 uMto about 10,000 mM and optionally in combination with other agents. Inother embodiments, the HDAC inhibitor is used at a concentration ofabout 10 uM to about 10000 mM and optionally in combination with otheragents. In other embodiments, the HDAC inhibitor is used at aconcentration of about 100 uM to about 1000 mM and optionally incombination with other agents. In other embodiments, the HDAC inhibitoris used at a concentration of about 1 uM to about 10 uM and optionallyin combination with other agents. In other embodiments, the HDACinhibitor is used at a concentration of about 10 uM to about 100 uM andoptionally in combination with other agents. In other embodiments, theHDAC inhibitor is used at a concentration of about 100 uM to about 1000uM and optionally in combination with other agents. In otherembodiments, the HDAC inhibitor is used at a concentration of about 1000uM to about 10 mM and optionally in combination with other agents. Inother embodiments, the HDAC inhibitor is used at a concentration ofabout 10 mM to about 100 mM and optionally in combination with otheragents. In other embodiments, the HDAC inhibitor is used at aconcentration of about 100 mM to about 1000 mM and optionally incombination with other agents. In other embodiments, the HDAC inhibitoris used at a concentration of about 1000 mM to about 10,000 mM andoptionally in combination with other agents.

In some embodiments of the compositions described herein, the HDACinhibitor is Valproic Acid used at a concentration of about 10 uM toabout 100,000 mM and optionally in combination with other agents. Inother embodiments, the HDAC inhibitor is Valproic Acid used at aconcentration of about 1 mM to about 10,000 mM and optionally incombination with other agents. In other embodiments, the HDAC inhibitoris Valproic Acid used at a concentration of about 10 mM to about 10,000mM and optionally in combination with other agents. In otherembodiments, the HDAC inhibitor is Valproic Acid used at a concentrationof about 100 mM to about 10,000 mM and optionally in combination withother agents. In other embodiments, the HDAC inhibitor is Valproic Acidused at a concentration of about 200 mM to about 2,000 mM and optionallyin combination with other agents. In other embodiments, the HDACinhibitor is Valproic Acid used at a concentration of about 1,000 mM andoptionally in combination with other agents.

In some embodiments of the compositions described herein, the Wntagonist, GSK-3 alpha inhibitor, or GSK-3 beta inhibitor is used at aconcentration ratio of about 0.01 to about 1,000,000-fold the activityin Effective Stemness Driver Concentration and optionally in combinationwith other agents. In other embodiments, the Wnt agonist, GSK-3 alphainhibitor, or GSK-3 beta inhibitor is used at a concentration ratio ofabout 0.1 to about 100,000-fold the Effective Stemness DriverConcentration and optionally in combination with other agents. In otherembodiments, the Wnt agonist, GSK-3 alpha inhibitor, or GSK-3 betainhibitor is used at a concentration ratio of about 1 to about10,000-fold the Effective Stemness Driver Concentration and optionallyin combination with other agents. In other embodiments, the Wnt agonist,GSK-3 alpha inhibitor, or GSK-3 beta inhibitor is used at aconcentration ratio of about 100 to about 5000-fold the EffectiveStemness Driver Concentration and optionally in combination with otheragents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitor, orGSK-3 beta inhibitor is used at a concentration ratio of about 50 toabout 2000-fold the Effective Stemness Driver Concentration andoptionally in combination with other agents. In other embodiments, theWnt agonist, GSK-3 alpha inhibitor, or GSK-3 beta inhibitor is used at aconcentration ratio of about 100 to about 1000-fold the EffectiveStemness Driver Concentration and optionally in combination with otheragents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitor, orGSK-3 beta inhibitor is used at a concentration ratio of about1,000-fold the Effective Stemness Driver Concentration and optionally incombination with other agents.

In some embodiments of the compositions described herein, the HDACinhibitor is used at a concentration ratio of about 0.1 to about1,000,000-fold the Effective Concentration and optionally in combinationwith other agents. In other embodiments, the HDAC inhibitor is used at aconcentration ratio of about 1 to about 100,000-fold the EffectiveConcentration and optionally in combination with other agents. In otherembodiments, the HDAC inhibitor is used at a concentration ratio ofabout 10 to about 10,000-fold the Effective Concentration and optionallyin combination with other agents. In other embodiments, the HDACinhibitor is used at a concentration ratio of about 100 to about1000-fold the Effective Concentration and optionally in combination withother agents. In other embodiments, the HDAC inhibitor is used at aconcentration ratio of about 1,000-fold the Effective Concentration andoptionally in combination with other agents.

In some embodiments of the compositions described herein, the Wntagonist, GSK-3 alpha inhibitors or GSK-3 beta inhibitor is used at aconcentration of about 0.01 nM to about 1,000 uM and optionally incombination with other agents. In other embodiments, the Wnt agonist,GSK-3 alpha inhibitors or GSK-3 beta inhibitor is used at aconcentration of about 0.1 nM to about 1,000 uM and optionally incombination with other agents. In other embodiments, the Wnt agonist,GSK-3 alpha inhibitors or GSK-3 beta inhibitor is used at aconcentration of about 1 nM to about 100 uM and optionally incombination with other agents. In other embodiments, the Wnt agonist,GSK-3 alpha inhibitors or GSK-3 beta inhibitor is used at aconcentration of about 10 nM to about 10 uM and optionally incombination with other agents. In other embodiments, the Wnt agonist,GSK-3 alpha inhibitors or GSK-3 beta inhibitor is used at aconcentration of about 1 nM to about 10 nM and optionally in combinationwith other agents. In other embodiments, the Wnt agonist, GSK-3 alphainhibitors or GSK-3 beta inhibitor is used at a concentration of about10 nM to about 100 nM and optionally in combination with other agents.In other embodiments, the Wnt agonist, GSK-3 alpha inhibitors or GSK-3beta inhibitor is used at a concentration of about 100 nM to about 1000nM and optionally in combination with other agents. In otherembodiments, the Wnt agonist, GSK-3 alpha inhibitors or GSK-3 betainhibitor is used at a concentration of about 1 uM to about 10 uM andoptionally in combination with other agents. In other embodiments, theWnt agonist, GSK-3 alpha inhibitors or GSK-3 beta inhibitor is used at aconcentration of about 10 uM to about 100 uM and optionally incombination with other agents.

In some embodiments of the compositions described herein, the GSK-3inhibitor is CHIR99021 used at a concentration of about 1 nM to about1,000 uM and optionally in combination with other agents. In otherembodiments, the GSK-3 inhibitor is CHIR99021 used at a concentration ofabout 10 nM to about 100 uM and optionally in combination with otheragents. In other embodiments, the GSK-3 inhibitor is CHIR99021 used at aconcentration of about 100 nM to about 100 uM and optionally incombination with other agents. In other embodiments, the GSK-3 inhibitoris CHIR99021 used at a concentration of about 1 uM to about 10 uM andoptionally in combination with other agents.

In some embodiments of the compositions described herein, the GSK-3inhibitor is LY2090314 used at a concentration of about 0.01 nM to about1,000 uM and optionally in combination with other agents. In otherembodiments, the GSK-3 inhibitor is LY2090314 used at a concentration ofabout 0.1 nM to about 10 uM and optionally in combination with otheragents. In other embodiments, the GSK-3 inhibitor is LY2090314 used at aconcentration of about 1 nM to about 1 uM and optionally in combinationwith other agents. In other embodiments, the GSK-3 inhibitor isLY2090314 used at a concentration of about 1 nM to about 100 nM andoptionally in combination with other agents. In other embodiments, theGSK-3 inhibitor is LY2090314 used at a concentration of about 10 nM andoptionally in combination with other agents.

In some embodiments of the compositions described herein, the GSK-3inhibitor is GSK inhibitor XXII used at a concentration of about 0.1 nMto about 1,000 uM and optionally in combination with other agents. Inother embodiments, the GSK-3 inhibitor is GSK inhibitor XXII used at aconcentration of about 1 nM to about 100 uM and optionally incombination with other agents. In other embodiments, the GSK-3 inhibitoris GSK inhibitor XXII used at a concentration of about 10 nM to about 10uM and optionally in combination with other agents. In otherembodiments, the GSK-3 inhibitor is GSK inhibitor XXII used at aconcentration of about 100 nM to about 1 uM and optionally incombination with other agents.

In some embodiments of the compositions described herein, the GSK-3inhibitor is AZD 1080 used at a concentration of about 1 nM to about1,000 uM and optionally in combination with other agents. In otherembodiments, the GSK-3 inhibitor is AZD 1080 used at a concentration ofabout 10 nM to about 1,000 uM and optionally in combination with otheragents. In other embodiments, the GSK-3 inhibitor is AZD 1080 used at aconcentration of about 100 nM to about 100 uM and optionally incombination with other agents. In other embodiments, the GSK-3 inhibitoris AZD 1080 used at a concentration of about 1 uM to about 10 uM andoptionally in combination with other agents.

In some embodiments of the compositions described herein, the HDACinhibitor is used at a concentration of about 0.01 nM to about 100,000uM and optionally in combination with other agents. In otherembodiments, the HDAC inhibitor is used at a concentration of about 1 nMto about 10,000 uM and optionally in combination with other agents. Inother embodiments, the HDAC inhibitor is used at a concentration ofabout 10 nM to about 10,000 uM and optionally in combination with otheragents. In other embodiments, the HDAC inhibitor is used at aconcentration of about 100 nM to about 1000 uM and optionally incombination with other agents. In other embodiments, the HDAC inhibitoris used at a concentration of about 1 nM to about 10 nM and optionallyin combination with other agents. In other embodiments, the HDACinhibitor is used at a concentration of about 10 nM to about 100 nM andoptionally in combination with other agents. In other embodiments, theHDAC inhibitor is used at a concentration of about 100 nM to about 1000nM and optionally in combination with other agents. In otherembodiments, the HDAC inhibitor is used at a concentration of about 1 uMto about 10 uM and optionally in combination with other agents. In otherembodiments, the HDAC inhibitor is used at a concentration of about 10uM to about 100 uM and optionally in combination with other agents. Inother embodiments, the HDAC inhibitor is used at a concentration ofabout 100 uM to about 1000 uM and optionally in combination with otheragents. In other embodiments, the HDAC inhibitor is used at aconcentration of about 1,000 uM to about 10,000 uM and optionally incombination with other agents.

In some embodiments of the compositions described herein, the HDACinhibitor is Valproic Acid used at a concentration of about 10 nM toabout 100,000 uM and optionally in combination with other agents. Inother embodiments, the HDAC inhibitor is Valproic Acid used at aconcentration of about 1 uM to about 10,000 uM and optionally incombination with other agents. In other embodiments, the HDAC inhibitoris Valproic Acid used at a concentration of about 10 uM to about 10,000uM and optionally in combination with other agents. In otherembodiments, the HDAC inhibitor is Valproic Acid used at a concentrationof about 100 uM to about 10,000 uM and optionally in combination withother agents. In other embodiments, the HDAC inhibitor is Valproic Acidused at a concentration of about 200 uM to about 2,000 uM and optionallyin combination with other agents. In other embodiments, the HDACinhibitor is Valproic Acid used at a concentration of about 1,000 uM andoptionally in combination with other agents.

Administration

The membrane of the round or oval window is the biological barrier tothe inner ear space and represents the major obstacle for the localtreatment of hearing impairment. The administered drug must overcomethis membrane to reach the inner ear space. The drug can operatively(e.g., injection through the tympanic membrane) be placed locally to theround or oval window membrane and can then penetrate through the roundor oval window membrane. Substances that penetrate the round or ovalwindow typically distribute in the perilymph and thus reach the haircells and supporting cells.

In certain embodiments, pharmaceutical compositions are adapted toadminister the drug locally to the round or oval window membrane. Thepharmaceutical compositions may also contain a membrane penetrationenhancer, which supports the passage of the agents mentioned hereinthrough the round or oval window membrane. Accordingly, liquid, gel orfoam compositions may be used. It is also possible to apply the activeingredient orally or to employ a combination of delivery approaches.

Intratympanic (IT) delivery of drugs to the ear is increasingly used forboth clinical and research purposes. Some groups have applied drugs in asustained manner using microcatheters and microwicks, while the majorityhave applied them as single or as repeated IT injections (up to 8injections over periods of up to 2 weeks)8.

Intratympanically applied drugs are thought to enter the fluids of theinner ear primarily by crossing the round or oval window (RW) membrane.Calculations show that a major factor controlling both the amount ofdrug entering the ear and the distribution of drug along the length ofthe ear is the duration the drug remains in the middle ear space.Single, ‘one-shot’ applications or applications of aqueous solutions forfew hours' duration result in steep drug gradients for the appliedsubstance along the length of the cochlea and rapidly decliningconcentration in the basal turn of the cochlea as the drug subsequentlybecomes distributed throughout the ear.

Other injection approaches include by osmotic pump, or, by combinationwith implanted biomaterial, by injection or infusion. Biomaterials thatcan aid in controlling release kinetics and distribution of drug includehydrogel materials, degradable materials. One class of materials that isused includes in situ gelling materials. All potential materials andmethodologies mentioned in these references are included herein byreference (Almeida H, Amaral M H, Lobao P, Lobo J M. In situ gellingsystems: a strategy to improve the bioavailability of ophthalmicpharmaceutical compositions. Drug Discovery Today 2014; 19:400-12; WiseA K, Gillespie L N. Drug delivery to the inner ear. Journal of NeuralEngineering 2012; 9:065002; Surovtseva E V, Johnston A H, Zhang W, etal. Prestin binding peptides as ligands for targeted polymersomemediated drug delivery to outer hair cells in the inner ear.International Journal of Pharmaceutics 2012; 424:121-7; Roy S, GlueckertR, Johnston A H, et al. Strategies for drug delivery to the human innerear by multifunctional nanoparticles. Nanomedicine 2012; 7:55-63; RiveraT, Sanz L, Camarero G, Varela-Nieto I. Drug delivery to the inner ear:strategies and their therapeutic implications for sensorineural hearingloss. Current Drug Delivery 2012; 9:231-42; Pararas E E, Borkholder D A,Borenstein J T. Microsystems technologies for drug delivery to the innerear. Advanced drug delivery reviews 2012; 64:1650-60; Li M L, Lee L C,Cheng Y R, et al. A novel aerosol-mediated drug delivery system forinner ear therapy: intratympanic aerosol methylprednisolone canattenuate acoustic trauma. IEEE Transactions on Biomedical Engineering2013; 60:2450-60; Lajud S A, Han Z, Chi F L, et al. A regulated deliverysystem for inner ear drug application. Journal of controlled release:official journal of the Controlled Release Society 2013; 166:268-76; KimD K, Park S N, Park K H, et al. Development of a drug delivery systemfor the inner ear using poly(amino acid)-based nanoparticles. Drugdelivery 2014; Kanzaki S, Fujioka M, Yasuda A, et al., PloS ONE 2012;7:e48480; Engleder E, Honeder C, Klobasa J, Wirth M, Arnoldner C, GaborF. Preclinical evaluation of thermoreversible triamcinolone acetonidehydrogels for drug delivery to the inner ear. International Journal ofPharmaceutics 2014; 471:297-302; Bohl A, Rohm H W, Ceschi P, et al.Development of a specially tailored local drug delivery system for theprevention of fibrosis after insertion of cochlear implants into theinner ear. Journal of Materials Science: Materials in Medicine 2012;23:2151-62; Hoskison E, Daniel M, Al-Zahid S, Shakesheff K M, Bayston R,Birchall J P. Drug delivery to the ear. Therapeutic Delivery 2013;4:115-24; Staecker H, Rodgers B., Expert Opin Drug Deliv 2013;10:639-50; Pritz C O, Dudas J, Rask-Andersen H, Schrott-Fischer A,Glueckert R. Nanomedicine strategies for drug delivery to the ear.Nanomedicine 2013; 8:1155-72), which are included herein by reference intheir entirety. Other materials include collagen or other naturalmaterials including fibrin, gelatin, and decellularized tissues. Gelfoammay also be suitable.

Delivery may also be enhanced via alternate means including but notlimited to agents added to the delivered composition such as penetrationenhancers, or could be through devices via ultrasound, electroporation,or high speed jet.

Methods described herein can also be used for inner ear cell types thatmay be produced using a variety of methods know to those skilled in theart including those cell types described in PCT Application No.WO2012103012 A1.

With regard to human and veterinary treatment, the amount of aparticular agent(s) that is administered may be dependent on a varietyof factors, including the disorder being treated and the severity of thedisorder; activity of the specific agent(s) employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific agent(s) employed; the duration of the treatment; drugs used incombination or coincidental with the specific agent(s) employed; thejudgment of the prescribing physician or veterinarian; and like factorsknown in the medical and veterinary arts.

The agents described herein may be administered in a therapeuticallyeffective amount to a subject in need of treatment. Administration ofcompositions described herein can be via any of suitable route ofadministration, particularly by intratympanically. Other routes includeingestion, or alternatively parenterally, for example intravenously,intra-arterially, intraperitoneally, intrathecally, intraventricularly,intraurethrally, intrasternally, intracranially, intramuscularly,intranasally, subcutaneously, sublingually, transdermally, or byinhalation or insufflations, or topical by ear instillation forabsorption through the skin of the ear canal and membranes of theeardrum. Such administration may be as a single or multiple oral doses,defined number of ear drops, or a bolus injection, multiple injections,or as a short- or long-duration infusion. Implantable devices (e.g.,implantable infusion pumps) may also be employed for the periodicparenteral delivery over time of equivalent or varying dosages of theparticular composition. For such parenteral administration, thecomposition s are formulated as a sterile solution in water or anothersuitable solvent or mixture of solvents. The solution may contain othersubstances such as salts, sugars (particularly glucose or mannitol), tomake the solution isotonic with blood, buffering agents such as acetic,citric, and/or phosphoric acids and their sodium salts, andpreservatives. The preparation of suitable and sterile parenteralcompositions is described in detail in the section entitled“Compositions” above.

Compositions described herein can be administered by a number of methodssufficient to deliver the composition to the inner ear. Delivering acomposition to the inner ear includes administering the composition tothe middle ear, such that the composition may diffuse across the roundor oval window to the inner ear and administering a composition to theinner ear by direct injection through the round or oval window membrane.Such methods include, but are not limited to auricular administration,by transtympanic wicks or catheters, or parenteral administration, forexample, by intraauricular, transtympanic, or intracochlear injection.

In particular embodiments, the compositions and compositions of thedisclosure are locally administered, meaning that they are notadministered systemically.

In one embodiment, a syringe and needle apparatus is used to administercompositions to a subject using auricular administration. A suitablysized needle is used to pierce the tympanic membrane and a wick orcatheter comprising the composition is inserted through the piercedtympanic membrane and into the middle ear of the subject. The device maybe inserted such that it is in contact with the round or oval window orimmediately adjacent to the round or oval window. Exemplary devices usedfor auricular administration include, but are not limited to,transtympanic wicks, transtympanic catheters, round or oval windowmicrocatheters (small catheters that deliver medicine to the round oroval window), and Silverstein Microwicks™ (small tube with a “wick”through the tube to the round or oval window, allowing regulation bysubject or medical professional).

In another embodiment, a syringe and needle apparatus is used toadminister compositions to a subject using transtympanic injection,injection behind the tympanic membrane into the middle and/or inner ear.The composition may be administered directly onto the round or ovalwindow membrane via transtympanic injection or may be administereddirectly to the cochlea via intracochlear injection or directly to thevestibular organs via intravestibular injection.

In some embodiments, the delivery device is an apparatus designed foradministration of compositions to the middle and/or inner ear. By way ofexample only: GYRUS Medical Gmbh offers micro-otoscopes forvisualization of and drug delivery to the round or oval window niche;Arenberg has described a medical treatment device to deliver fluids toinner ear structures in U.S. Pat. Nos. 5,421,818; 5,474,529; and5,476,446, each of which is incorporated by reference herein for suchdisclosure. U.S. patent application Ser. No. 08/874,208, which isincorporated herein by reference for such disclosure, describes asurgical method for implanting a fluid transfer conduit to delivercompositions to the inner ear. U.S. Patent Application Publication2007/0167918, which is incorporated herein by reference for suchdisclosure, further describes a combined otic aspirator and medicationdispenser for transtympanic fluid sampling and medicament application.

In some embodiments, a composition disclosed herein is administered to asubject in need thereof once. In some embodiments, a compositiondisclosed herein is administered to a subject in need thereof more thanonce. In some embodiments, a first administration of a compositiondisclosed herein is followed by a second, third, fourth, or fifthadministration of a composition disclosed herein.

The number of times a composition is administered to a subject in needthereof depends on the discretion of a medical professional, thedisorder, the severity of the disorder, and the subject's response tothe composition. In some embodiments, a composition disclosed herein isadministered once to a subject in need thereof with a mild acutecondition. In some embodiments, a composition disclosed herein isadministered more than once to a subject in need thereof with a moderateor severe acute condition. In the case wherein the subject's conditiondoes not improve, upon the doctor's discretion the composition may beadministered chronically, that is, for an extended period of time,including throughout the duration of the subject's life in order toameliorate or otherwise control or limit the symptoms of the subject'sdisease or condition.

In the case wherein the subject's status does improve, upon the doctor'sdiscretion the composition may administered continuously; alternatively,the dose of drug being administered may be temporarily reduced ortemporarily suspended for a certain length of time (i.e., a “drugholiday”). The length of the drug holiday varies between 2 days and 1year, including by way of example only, 2 days, 3 days, 4 days, 5 days,6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250days, 280 days, 300 days, 320 days, 350 days, and 365 days. The dosereduction during a drug holiday may be from 10%-100%, including by wayof example only 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%.

Once the subject's hearing and/or balance has improved, a maintenancedose can be administered, if necessary. Subsequently, the dosage or thefrequency of administration, or both, is optionally reduced, as afunction of the symptoms, to a level at which the improved disease,disorder or condition is retained. In certain embodiments, subjectsrequire intermittent treatment on a long-term basis upon any recurrenceof symptoms

EXAMPLES Example 1: Effects of Poloxamer and Valproate on CHIR99021Dihydrochloride (diHCl)

A 23.75% (w/v) poloxamer solution was prepared in 1×PBS containing 1M(166.2 mg/ml) Valproic acid sodium salt, 5% (v/v) DMSO, and 2.77 mM(1.39 mg/ml) CHIR99021 freebase. The poloxamer was fully soluble in thesolutions at this concentration and the solution retained fluidity at 4°C. as judged by passage through a micropipette with a 100 uM opening.The solution also retained poloxamer polymerization capacity at 37° C.as judged by gelling of 10 ul of solution pipetted onto coverglasswarmed to 37° C. in less than 20 seconds. The final pH of this solutionis around 9.

Example 2: Effects on Solubility at Various Concentrations of Poloxamer

P407 solubility was studied at 16.7%, 20%, 23.1%, 25.9%, 28.7%, 33.3%,37.5%, 41.2%, and 50% w/w in 1M Valproic acid sodium salt and 5%dimethylsulfoxide (DMSO). Valproic acid sodium salt (NaVal) stocksolution was prepared by dissolving 16.62 g Valproic acid sodiumsalt+5.0 g DMSO+78.37 g Water. Poloxamer 407 (P407) was weighed andadded to containers containing 10 g each of the 1M Valproic acid sodiumsalt+5% w/w DMSO solution. The solutions were stored overnight inrefrigerator at 0-2° C. and observed for any undissolved poloxamer.Poloxamer levels of up to 28.6% are acceptable when formulated with 1 Mvalproic acid sodium salt and 5% dimethyl sulfoxide.

Results

Samples at a concentration of 16.7% to 23.1% w/w P407 were clear andfluid in cold conditions while solutions at a concentration of 25.9% to28.6% w/w P407 were clear but viscous. (FIGS. 1 and 2) Concentrationsabove 33.3% w/w P407 remained undissolved and formed a thick swollenmass. The results demonstrated that poloxamer levels of up to 28.6% areacceptable when formulated with 1 M valproic acid sodium salt and 5%dimethyl sulfoxide. Table 1 below shows the results and observations ofthe samples at various concentrations of P407.

TABLE 1 % P407 18+ Hours 40+ Hours Composition (w/w) ObservationsObservations 2 g P407; 10 g 1M NaVal 16.70% Clear, Clear, and 5% DMSOStock dissolved dissolved solution solution 4 g P407; 10 g 1M NaVal28.60% Almost clear, Clear, and 5% DMSO Stock small white dissolved,globs viscous gel 5 g P407; 10 g 1M NaVal 33.30% Slightly swollenSwollen white and 5% DMSO Stock white glob glob 6 g P407; 10 g 1M NaVal37.50% Swollen white Swollen white and 5% DMSO Stock glob glob 7 g P407;10 g 1M NaVal 41.20% Swollen white Swollen white and 5% DMSO Stock globglob 10 g P407; 10 g 1M NaVal   50% Extremely Extremely and 5% DMSOStock swollen swollen white glob white glob 2.5 g P407; 10 g 1M NaVal  20% n/a Clear, and 5% DMSO Stock dissolved solution 3 g P407; 10 g 1MNaVal 23.10% n/a Clear, and 5% DMSO Stock dissolved solution 3.5 g P407;10 g 1M NaVal 25.90% n/a Clear, and 5% DMSO Stock dissolved, slightlyviscous solution

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Methods and materials are describedherein for use in the present invention; other, suitable methods andmaterials known in the art can also be used. The materials, methods, andexamples are illustrative only and not intended to be limiting. Suchembodiments are also within the scope of the following claims. Therecitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof. Theteachings of all patents, published applications and references citedherein are incorporated by reference in their entirety. While thisinvention has been particularly shown and described with references toexample embodiments thereof, it will be understood by those skilled inthe art that various changes in form and details may be made thereinwithout departing from the scope of the invention encompassed by theappended claims

1-520. (canceled)
 521. A pharmaceutical composition comprising: a) a2-pyrimidinylaminoethylamino-2-pyridine compound; b) Poloxamer 407; andc) a carboxylic acid containing compound, or a pharmaceuticallyacceptable salt thereof; wherein the pharmaceutical composition has agelation temperature of greater than 10° C.
 522. The pharmaceuticalcomposition of claim 521, wherein the2-pyrimidinylaminoethylamino-2-pyridine compound is a2,4-dichlorophenyl-5-(1H-imidazol-2-yl)-2-pyrimidinylaminoethylamino-2-pyridinecompound.
 523. The pharmaceutical composition of claim 521, wherein the2-pyrimidinylaminoethylamino-2-pyridine compound is6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile,having the structure:


524. The pharmaceutical composition of claim 521, wherein the Poloxamer407 is present at a concentration of great than or equal to 21 wt %.525. The pharmaceutical composition of claim 521, wherein the Poloxamer407 is present at a concentration of about 23 wt % to about 29 wt %.526. The pharmaceutical composition of claim 521, wherein the carboxylicacid containing compound is a substituted or unsubstituted C₅-C₉straight or branched chain alkyl-CO₂H.
 527. The pharmaceuticalcomposition of claim 521, wherein the carboxylic acid containingcompound is valproic acid.
 528. The pharmaceutical composition of claim521, wherein the pharmaceutical composition has a gelation temperatureof about 11° C. and about 32° C.
 529. A pharmaceutical compositioncomprising: a) a 2-pyrimidinylaminoethylamino-2-pyridine compound; b)Poloxamer 407, wherein the Poloxamer 407 is present at a concentrationof greater than or equal to 21 wt %; and c) a carboxylic acid containingcompound, or a pharmaceutically acceptable salt thereof.
 530. Thepharmaceutical composition of claim 529, wherein the pharmaceuticalcomposition has a gelation temperature of greater than 10° C.
 531. Thepharmaceutical composition of claim 529, wherein the pharmaceuticalcomposition has a gelation temperature of about 11° C. and about 32° C.532. The pharmaceutical composition of claim 529, wherein the2-pyrimidinylaminoethylamino-2-pyridine compound is a2,4-dichlorophenyl-5-(1H-imidazol-2-yl)-2-pyrimidinylaminoethylamino-2-pyridinecompound.
 533. The pharmaceutical composition of claim 529, wherein the2-pyrimidinylaminoethylamino-2-pyridine compound is6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile,having the structure:


534. The pharmaceutical composition of claim 529, wherein the Poloxamer407 is present at a concentration of about 23 wt % to about 29 wt %.535. The pharmaceutical composition of claim 529, wherein the carboxylicacid containing compound is a substituted or unsubstituted C₅-C₉straight or branched chain alkyl-CO₂H.
 536. The pharmaceuticalcomposition of claim 529, wherein the carboxylic acid containingcompound is valproic acid.